Method of attenuating porcine pseudorabies virus, attenuated strains of porcine pseudorabies virus, vaccine composition and use thereof

1. The well-grown Marc-145 cells digested with trypsin, were inoculated in cell culture flasks and then cultured at 36° C.˜38° C. with cell growth medium (pH was adjusted to 7.0˜8.0) containing 90%˜97% (V/V) DMEM culture medium and 3%˜10% (V/V) fetal bovine serum, to form a proper monolayer for inoculation with virus.

2. The PRV HN1201 strain was inoculated into the above well-grown monolayer of subcultured cells, and the cells continued to be cultured at 36° C.˜38° C. with cell maintenance medium (pH was adjusted to 7.1˜7.5) containing 95%˜99% (V/V) DMEM and 1%˜5% (V/V) fetal bovine serum. After 40 h˜48 h, the cell medium containing viruses, i.e. the pseudorabies virus strain adapted to mammalian cells, was harvested when the cytopathic effect of cells reached 80%, as the virus seed for continued passage. Different passages of virus harvested, P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P15, P20, P30, P50, P70, P90, P110, P130, P150 and P200, was sequenced respectively, with the results indicating no deficiency of genes for each passage.

3. The well-grown DF-1 cells digested with trypsin, were inoculated in cell culture flasks and then cultured at 36° C.˜38° C. with cell growth medium (pH was adjusted to 7.0˜8.0) containing 90%˜97% (V/V) DMEM and 3%˜10% (V/V) fetal bovine serum, to form a proper monolayer for inoculation with virus.

4. Different passages of the pseudorabies virus strain adapted to mammalian cells harvested in the step 2, was inoculated into the above well-grown monolayer of subcultured DF-1 cells obtained from the step 3, and continued to be cultured at 36° C.˜38° C. with cell maintenance medium (pH was adjusted to 7.1˜7.5) containing 95%˜99% (V/V) DMEM and 1%˜5% (V/V) fetal bovine serum. After 40 h˜48 h, the cell medium containing viruses was harvested respectively when the cytopathic effect of cells reached 80%, i.e. P1-1, P2-1, P3-1, P4-1, P5-1, P6-1, P7-1, P8-1, P9-1, P10-1, P15-1, P20-1, P30-1, P50-1, P70-1, P90-1, P110-1, P130-1, P150-1, P200-1. Each passage of viruses harvested was sequenced respectively, with the results indicating that there was no deficiency of genes for P1-1, P2-1, P3-1, P4-1, while P5-1, P6-1, P7-1, P8-1, P9-1, P10-1, P15-1, P20-1, P30-1, P50-1, P70-1, P90-1, P110-1, P130-1, P150-1 and P200-1 all has the deficiency of genes, in which each has continuous deficiency of 3455 bp started from the site of the 890 th nucleotide of gI gene.

In order to verify if it is related to passage times in DF-1 that there was no deficiency of genes for P1-1, P2-1, P3-1 and P4-1, such serial passage continued and P1-2, P1-3, P1-4, P1-5, P1-6, P1-7, P1-8, P1-9, P1-10, P2-2, P2-3, P2-4, P2-5, P2-6, P2-7, P2-8, P2-9, P2-10, P3-2, P3-3, P3-4, P3-5, P3-6, P3-7, P3-8, P3-9, P3-10, P4-2, P4-3, P4-4, P4-5, P4-6, P4-7, P4-8, P4-9 and P4-10 was harvested respectively. Each harvested virus was sequenced respectively, with the results indicating no deficiency of genes for each passage. It showed that in the case where PRV was passaged four times or less in Marc-145 cells, the occurrence of deficiency of genes was not related to passage times in DF-1, but to passage times of adaption in Marc-145 cells.

In order to verify the stability of continued passage in Df-1 for the viruses with deficiency of genes, P5-1, P6-1, P7-1, P8-1, P9-1, P10-1, P15-1, P20-1, P30-1, P50-1, P70-1, P90-1, P110-1, P130-1, P150-1, P200-1 was continued to be passaged, resulted to the harvest of P5-2, P5-3, P5-4, P5-5, P5-6, P5-7, P5-8, P5-9, P5-10, P6-2, P6-3, P6-4, P6-5, P6-6, P6-7, P6-8, P6-9, P6-10, P7-2, P7-3, P7-4, P7-5, P7-6, P7-7, P7-8, P7-9, P7-10, P8-2, P8-3, P8-4, P8-5, P8-6, P8-7, P8-8, P8-9, P8-10, P9-2, P9-3, P9-4, P9-5, P9-6, P9-7, P9-8, P9-9, P9-10, P10-2, P10-3, P10-4, P10-5, P10-6, P10-7, P10-8, P10-9, P10-10, P15-2, P15-3, P15-4, P15-5, P15-6, P15-7, P15-8, P15- 9, P15-10, P20-2, P20-3, P20-4, P20-5, P20-6, P20-7, P20-8, P20-9, P20-10, P30-2, P30-3, P30- 4, P30-5, P30-6, P30-7, P30-8, P30-9, P30-10, P50-2, P50-3, P50-4, P50-5, P50-6, P50-7, P50- 8, P50-9, P50-10, P70-2, P70-3, P70-4, P70-5, P70-6, P70-7, P70-8, P70-9, P70-10, P90-2, P90- 3, P90-4, P90-5, P90-6, P90-7, P90-8, P90-9, P90-10, P110-2, P110-3, P110-4, P110-5, P110-6, P110-7, P110-8, P110-9, P110-10, P130-2, P130-3, P130-4, P130-5, P130-6, P130-7, P130-8, P130-9, P130-10, P150-2, P150-3, P150-4, P150-5, P150-6, P150-7, P150-8, P150-9, P150-10, P200-2, P200-3, P200-4, P200-5, P200-6, P200-7, P200-8, P200-9 and P200-10. Each passage of harvested viruses was sequenced respectively, with the results indicating that there was no change for the occurrence of deficiency of genes for each passage, in which each always has continuous deficiency of 3455 bp started from the site of the 890 th nucleotide of gI gene, indicating a stable passage in DF-1 for the PRV gene-deleted strain.

The attenuated strain of PRV, P30-10 was named PRV HN1201-R strain.

1. Pathogenicity Test of the Virus

15 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 3 groups (A, B and blank control groups), each with 5 piglets. The grouping and challenging conditions are shown in Table 1.

Piglets were observed for 28 days after inoculation of virus, while the temperature of piglets was determined daily, and clinical signs and death status were observed. The specific results are shown in Table 2.

It showed in the results that inoculation with PRV HN1201 strain in 7-day-old piglets could lead to death with a morality rate of 100% (5/5) of inoculated piglets, while PRV HN1201-R strain displayed a significant reduction of virulence, only causing increased body temperature of two pigs, without any other clinical signs, or any change of tissues or organs obtained from the necropsy.

Through the pathogenicity test it indicated that compared with the parent virulent strain, i.e. PRV HN1201 strain, PRV HN1201-R strain displayed a significant reduction of pathogenicity, and was an attenuated virus strain.

Meanwhile, in order to verify the stability of pathogenicity of different passages of PRV HN1201-R strain, a group of piglets (5 piglets) at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1201-R strain, respectively, and each was inoculated with 1 ml (10 7.0 TCID 50 /ml) by intranasal instillation. Another five piglets were used as the control group. The clinical manifestations of piglets were observed and recorded daily until 28 days after inoculation of virus.

It showed in the results that, from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1201-R strain, PRV HN1201-R strain displayed a significant reduction of virulence, only causing increased body temperature of 1˜2 pigs/group, without any other clinical signs, or any change of tissues or organs obtained from the necropsy.

Through the pathogenicity test of different passages, it indicated that different passages of PRV HN1201-R strain all displayed lower virulence.

2. Immunogenicity Assay

On the 21 st day after immunization, all of the five piglets inoculated with PRV 1201-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1201. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 3.

The result indicated that all the piglets inoculated with PRV HN1201-R strain were healthy and alive, while all from the control group died.

According to the immunogenicity assay, the PRV HN1201-R strain can provide excellent protection against PRV HN1201 strain, showing excellent immunogenicity.

Meanwhile, in order to verify the stability of immunogenicity of different passages of PRV HN1201-R strain, on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1201-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1201. After challenge, the body temperature of piglets was determined daily, and in the meanwhile clinical signs and death status were observed.

The result indicated that all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1201-R strain were alive, while all from the control group died.

According to the immunogenicity assay of different passages, different passages of PRV HN1201-R strain all can provide excellent protection against PRV HN1201 strain, showing excellent immunogenicity.

3. Reversion of Virulence Test of the Virus

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 5 groups, each with 6 piglets. 6 piglets of Group 1 which were negative for pseudorabies antigens and antibodies were inoculated with 10 7.0 TCID 50 /piglet of the cultures of PRV HN1201-R strain (the 1 st , 30 th , 60 th , 85 th , 110 th passages and the 1 st +30 th +60 th +85 th +110 th passages) respectively by intranasal instillation. On day 14, they were raised together with the 6 piglets of Group 2 which were negative for pseudorabies antigens and antibodies. 14 days later, the 6 piglets of Group 1 were drawn back, and again, the 6 piglets of Group 3, which were negative for pseudorabies antigens and antibodies, were raised together with the 6 piglets of Group 2 and so on. After 4 serial passages, all the drawn piglets were killed in order to observe if there were any pathological changes.

It showed in the result that no abnormal changes were found during the clinical observation and gross anatomy of 30 experimental piglets by the 4th serial passage of the co-habitation infection experiment, indicating that there was no reversion of virulence of this attenuated strain. Therefore, the safety of the vaccines can be ensured since the virus, after being inoculated into piglets, would not evolve into a virulent virus which is able to cause disease.

4. Genes Sequences Analysis

The genome amplification of the cultures of the 1 st passage to 110 th passage of PRV HN1201-R strain was accomplished by means of RT-PCR (The genomic DNA of culture of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acid sequence of the virus. The obtained amino acid sequence was compared with the amino acid sequence of the parent virulent strain, HN1201 strain via sequence analysis software, and the amino acids sequence of the virus was characterized.

It showed in the results that for the cultures of the 1 st passage to 110 th passage of PRV HN1201-R strain, each amino acid sequences encoded by the viral genes commonly has continuous deficiency of gI/gE/11K/28K genes (3455 bp in total), i.e. continuous deficiency of 3455 bp started from the site of the 890 th nucleotide of gI gene. The deleted sequence is as shown in SEQ No. 1.

It indicated that a common characteristic change of the amino acids sequences encoded by the viral genes of the culture of different passages of PRV HN1201-R strain might be the reason for the reduction of virulence of the parent virulent strain.

1. Proliferation of Virus

The virus seed of PRV HN1201-R strain prepared in Example 1 was diluted at 5×10 4 fold, and then inoculated into a monolayer of ST cell. After 1 h adhesion, 1000 ml of DMEM medium containing 2% fetal calf serum was added into ST cell, which was then placed at 37° C. in a roller bottle with a rotation speed of 6 rph. The cell medium containing viruses was harvested when the cytopathic effect of cells reached 80%; the viruses were harvested after 2 times of freezing-thawing the medium, and the virus titer was assessed. The virus solution was preserved at low temperature.

2. Preparation of a Protective Agent

40 g of sucrose and 8 g of gelatin was added into every 100 ml of deionized water, and the solution was autoclaved (under 121° C. for 30 min) after being fully melted.

3. Preparation of Vaccine

The virus solution prepared and preserved from above procedure was mixed with the protective agent prepared and preserved from above procedure at a volume ratio of 1:1 and the mixed virus solution was freeze-dried. The specific ratio of content of the vaccine is shown in Table 4.

15 9-day-old piglets which were negative for PRV antibodies and PRV antigens were randomly divided into 3 groups, each with 5 piglets, and the piglets were injected with the attenuated live PRV HN1201-R strain prepared in Example 3. The first group was inoculated with Vaccine 1, and the second group was inoculated with Vaccine 2, and the third group was the control group. The piglets were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1201 strain on day 21 after immunization. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 5.

The result indicated that immunizing piglets with the attenuated live vaccine of PRV HN1201-R strain prepared in example 3 can block virus infection (i.e. prevent occurrence of clinic signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 4 after challenge.

It has proven that the attenuated live vaccine of PRV HN1201-R strain in two experimental groups can provide excellent protection, showing excellent immune protection and safety; in the meanwhile it indicated that PRV strain which has a continuous deficiency of gI/gE/11K/28K genes (3455 bp in total) would still maintain excellent immunogenicity.

15 9-day-old piglets which were negative for PRV antibodies and PRV antigens were randomly divided into 3 groups, each with 5 piglets. The first group was inoculated with Vaccine 1, the attenuated live vaccine of HN1201-R strain prepared in Example 3; the second group was immunized with the live PRV vaccine, Bartha K-61 strain purchased from HIPRA, Spain, with Batch No. 42RH; and the third group is the blank control group. The piglets were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1201 strain on day 28 after immunization. After challenge, the body temperature of piglets was determined daily, and clinical signs and death status were observed as well. The detailed results are shown in Table 6.

The result indicated that immunizing piglets with the attenuated live vaccine of PRV HN1201-R strain prepared in Example 3 can block virus infection (i.e. prevent occurrence of clinic signs), and provide 100% (5/5) protection rate for piglets; all the piglets in the control group died by day 4 after challenge; whereas the commercial vaccines in the prior art cannot provide full protection for pigs.

It has proven that the attenuated live vaccine of PRV HN1201-R strain can provide excellent protection, showing better immune protection and safety than the commercial vaccines in the prior art.

PRV HN1202 strain and Fa strain were subcultured respectively according to the procedures in Example 1, so as to obtain their attenuated PRV strains, named PRV HN1202-R strain and Fa-R strain.

1. Pathogenicity Test of the Virus

20 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 4 groups (C, D, E and F), each with 5 piglets; another 10 piglets were used as the blank control group. The grouping and challenging conditions are shown in Table 7.

Piglets were observed for 28 days after inoculation of virus, while the temperature of piglets was measured daily, and clinical signs and death status were observed. The results are shown in Table 8.

It showed in the results that inoculation with PRV HN1202 strain in 7-day-old piglets could lead to death of 100% (5/5) of inoculated piglets, while PRV HN1202-R strain displayed a significant reduction of virulence, only causing increased body temperature of three pigs, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; inoculation with PRV Fa strain in 7-day-old piglets could lead to death of 100% (5/5) of inoculated piglets, while PRV Fa-R strain displayed a significant reduction of virulence, only causing increased body temperature of four pigs, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy.

Through the pathogenicity test it indicated that compared with the parent virulent strain, i.e. PRV HN1202 strain, PRV HN1202-R strain displayed a significant reduction of pathogenicity; and was an attenuated virus strain; compared with the parent virulent strain, i.e. PRV Fa strain, PRV Fa-R strain displayed a significant reduction of pathogenicity, and was an attenuated virus strain.

Meanwhile, in order to verify the stability of pathogenicity of different passages of PRV HN1202-R strain and Fa-R strain, a group (5) of piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with 1 ml (10 7.0 TCID 50 /ml) of the cultures of 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1202-R strain by intranasal instillation, respectively, and another five piglets were used as the blank control group; a group (5) of piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with 1 ml (10 7.0 TCID 50 /ml) of the cultures of the 1 st , 30 th , 85 th and 110 th passages of PRV Fa-R strain by intranasal instillation, respectively, and another five piglets were used as the control group. The clinical manifestations of piglets were observed and recorded daily until 28 days after inoculation of virus.

It showed in the results that, from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1202-R strain, PRV HN1202-R strain displayed a significant reduction of virulence, only causing increased body temperature of 2˜3 pigs/group, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1202-R strain, PRV Fa-R strain displayed a significant reduction of virulence, only causing increased body temperature of 3-4 pigs/group, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy.

Through the pathogenicity test of different passages, it indicated that different passages of PRV HN1202-R strain and Fa-R strain all displayed lower virulence.

2. Immunogenicity Assay

On the 21 st day after immunization, all the five piglets inoculated with PRV 1202-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV 1202 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 9.

On the 21 st day after immunization, all the five piglets inoculated with PRV Fa-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV Fa strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 9.

The result indicated that all the piglets inoculated with PRV HN1202-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with PRV Fa-R strain were healthy and alive, while all from the control group died.

According to the immunogenicity assay, the PRV HN1202-R strain can provide excellent protection against PRV HN1202 strain, showing excellent immunogenicity; the PRV Fa-R strain can provide excellent protection against PRV Fa strain, showing excellent immunogenicity.

Meanwhile, in order to verify the stability of immunogenicity of different passages of PRV HN1202-R strain and Fa-R strain, on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1202-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1202; after challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed; on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV Fa-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV Fa. After challenge, the body temperature of piglets was determined daily, and in the meanwhile clinical signs and death status were observed.

The result indicated that all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HN1202-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV Fa-R strain were healthy and alive, while all from the control group died.

According to the immunogenicity assay of different passages, the culture of different passages of PRV HN1202-R strain all can provide excellent protection against PRV HN1202 strain, showing excellent immunogenicity; the culture of different passages of PRV Fa-R strain all can provide excellent protection against PRV Fa strain, showing excellent immunogenicity.

3. Reversion of Virulence Test of the Virus

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 5 groups, each with 6 piglets. 6 piglets of Group 1 which were negative for pseudorabies antigens and antibodies were inoculated with 10 7.0 TCID 50 /piglet of the cultures of PRV HN1202-R (the 1 st , 30 th , 60 th , 85 th , 110 th passages and the 1 st +30 th +60 th +85 th +110 th passages) respectively by intranasal instillation. On day 14, they were raised together with the 6 piglets of Group 2 which were negative for pseudorabies antigens and antibodies. 14 days later, the 6 piglets of Group 1 were drawn back, and again, the 6 piglets of Group 3, which were negative for pseudorabies antigens and antibodies, were raised together with the 6 piglets of Group 2 and so on. After 4 continuous passages, all the drawn piglets were killed in order to observe if there were any pathological changes.

It showed in the result that no abnormal changes were found during the clinical observation and gross anatomy of 30 experimental piglets infected with HN1202-R strain and 30 experimental piglets infected with Fa-R strain, by the 4th serial passage of the cohabitation infection experiment, indicating that there was no reversion of virulence of the two attenuated strains. Therefore, the safety of the vaccines can be ensured since the viruses, after being inoculated into piglets, would not evolve into virulent viruses which are able to cause disease.

4. Gene Sequences Analysis

The genome amplification of the cultures of the 1 st passage to 110 th passage of PRV HN1202-R strain was accomplished by means of RT-PCR (The genomic DNA of culture of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acid sequence of the virus. The obtained amino acid sequence was compared with the amino acid sequence of the parent virulent strain, i.e. PRV HN1202 strain via sequence analysis software, and the amino acids sequence of the virus was characterized.

Meanwhile, the genome amplification of the cultures of the 1 st passage to 110 th passage of PRV Fa-R strain was accomplished by means of RT-PCR (The genomic DNA of culture of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acid sequence of the virus. The obtained amino acid sequence was compared with the amino acid sequence of the parent virulent strain, PRV Fa strain via software for sequence analysis, and the amino acids sequence of the virus was characterized.

It showed in the results that for the cultures of the 1 st passage to 110 th passage of PRV HN1202-R strain, the amino acids sequences encoded by each viral genes commonly have continuous deficiency of gI/gE/11K/28K genes (3455 bp in total), in which the deficiency site and size are totally the same as those of PRV HN1201-R strain; for the cultures of the 1 st passage to 110 th passage of PRV Fa-R strain, the amino acids sequences encoded by each viral genes commonly have continuous deficiency of gI/gE/11K/28K genes (3455 bp in total), in which the deficiency site and size are totally the same as those of PRV HN1201-R strain; compared with their parent virulent strains, each of PRV HN1202-R strain and PRV Fa-R strain has continuous deficiency of gI/gE/11K/28K genes (3455 bp in total).

It indicated that a common characteristic change of the amino acids sequences encoded by the viral genes of the cultures of the different passages of PRV HN1202-R strain as well as PRV Fa-R strain is consistent with that of the amino acids encoded by the viral genes of PRV HN1201-R strain, which is caused by the deficiency of genes encoding said amino acids sequences, showing the stability of the method of attenuating the PRV by passage according to the present invention, and in the meanwhile, further indicating that continuous deficiency of gI/gE/11K/28K genes (3455 bp in total) in PRV is the reason for the reduction of virulence of its parent virulent strain.

Example 8: Preparation of the Attenuated Live Vaccines of PRV HN1202-R Strain and Fa-R Strain

The attenuated live vaccines of PRV HN1202-R strain and Fa-R strain were prepared according to the procedure in Example 3. The specific ratios of contents of the vaccines are shown in Table 10.

Example 9: Immunogenicity Assay of the Attenuated Live Vaccine of PRV HN1202-R Strain and Fa-R Strain

20 9-day-old piglets which were negative for PRV antigens and antibodies and PRV antigens were randomly divided into 4 groups, each with 5 piglets, and the piglets were injected with the attenuated live vaccine of PRV HN1202-R strain and Fa-R strain prepared in Example 8. The first group was immunized with Vaccine 3, and the third group was immunized with Vaccine 4, and the second and fourth group was the control group. The piglets in the first and second groups were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1202 strain on day 21 after immunization, and those in the third and fourth groups were challenged with 1×10 7.0 TCID 50 /piglet of PRV Fa strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The specific results are shown in Table 11.

The result indicated that immunizing piglets with the attenuated live vaccine of PRV HN1202-R strain prepared in example 8 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge; immunizing piglets with the attenuated live vaccine of PRV Fa-R strain prepared in example 8 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge.

It has proven that the attenuated live vaccines of PRV HN1202-R strain and Fa-R strain can provide excellent protection, showing excellent immune protection and safety; meanwhile it indicated again that a continuous deficiency of gI/gE/11K/28K genes (3455 bp in total) from the PRV virus would have no effect on its immunogenicity.

Example 10: Construction of PRV HN1201-gI − /gE − /11K − /28K − Gene-Deleted Strain

In order to obtain the PRV HN1201-gI − /gE − /11K − /28K − gene-deleted strain, the gI/gE/11K/28K genes were knocked out through molecular cloning by means of homologous recombination. The detailed procedures are as follows:

The sequence at the front end of US7 and the sequence at the back section of US2 were amplified respectively, as the left and right homologous recombinant arms US7L and US2R, with the genomic DNA of PRV HN1201 strain as the template, and US7-LP1/US7-LP2 and US2-RP1/US2-RP2 as the primers, in which there is a loxP site at each end of gIL and US2R. The transfer vector, pSKUS7-2-GFP with the loxP sites was constructed by use of pBluescript SK plasmid, with the green fluorescent protein GFP as the selectable marker.

The total DNA of PK-15 cells infected by PRV HN1201 strain was extracted via the DNAZol method, and the total DNA and the transfer vector, pSKUS7-2-GFP was co-transfected at the ratio of 10 μg:1 μg, into the PK-15 cells via the lipofectin-mediated transfection. The viruses were harvested when the cytopathic effect of cells reached 80%. After serial dilution, the harvested viruses were inoculated into a monolayer of PK-15 cells, to obtain the recombinant virus rPRV-US7-2 − /GFP + with GFP by means of plaque purification. 10 μg of the DNA of rPRV-US7-2 − /GFP + was added with 2.5 units of Cre recombinase and reacted for 1 h at 37° C.; the DNA was extracted to prepare DNA of rPRV-US7-2 − , which was then transfected into PK-15 cells. After plaques purification, the gene-deleted PRV strain containing no GFP, PRV HN1201-gI − /gE − /11K − /28K − gene-deleted strain was obtained.

Example 11: Preparation of the Attenuated Live Vaccines of PRV HN1201-gI − /gE − /11K − /28K − Gene-Deleted Strain

The attenuated live vaccines of PRV HN1201-gI − /gE − /11K − /28K − gene-deleted strain were prepared according to the procedure in Example 3. The specific ratios of content of the vaccine are shown in Table 12.

10 9-day-old piglets which were negative for PRV antigens and antibodies were randomly divided into 2 groups, each with 5 piglets, and the piglets were immunized with the attenuated live vaccine of PRV HN1201-gI − /gE − /11K − /28K − strain. The piglets were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1201 strain on day 21 after immunization. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The detailed results are shown in Table 13.

The results indicated that immunizing piglets with the attenuated live vaccine of PRV HN1201-gI − /gE − /11K − /28K − strain prepared by means of genetic engineering can block virus infection (i.e. prevent occurrence of clinical signs), and provide 80% (4/5) protection rate for piglets, while all the piglets in the blank control group died by day 4 after challenge.

It has proven that the attenuated live vaccine of PRV HN1201-gI − /gE − /11K − /28K − strain can provide excellent protection, showing excellent immune protection and safety; meanwhile it indicated again that a continuous deficiency of gI/gE/11K/28K genes (3455 bp in total) from the PRV virus would have no effect on its immunogenicity.

PRV PRV-ZJ01 strain, HeN1 strain and JS-2012 strain were subcultured to be attenuated respectively according to the procedures in Example 1, in order to obtain their attenuated PRV strains, named PRV-ZJ01-R strain, HeN1-R strain and JS-2012-R strain.

Example 13: Study of Biological Characteristics of PRV-ZJ01-R Strain, PRV HeN1-R Strain and PRV JS-2012-R Strain

1. Pathogenicity Test of the Virus

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 6 groups (G, H, I, J, L and M), each with 5 piglets; another 15 piglets were used as the blank control group. The grouping and challenging conditions are shown in Table 14.

Piglets were observed for 28 days after inoculation of virus, while the temperature of piglets was measured daily, and clinical signs and death status were observed. The specific results are shown in Table 15.

It showed in the results that inoculation with PRV PRV-ZJ01 strain into 7-day-old piglets could lead to death of 100% (5/5) of inoculated piglets, while PRV PRV-ZJ01-R strain displayed a significant reduction of virulence, only causing increased body temperature of four pigs, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; inoculation with PRV HeN1 strain into 7-day-old piglets could lead to death of 100% (5/5) of inoculated piglets, while PRV HeN1-R strain displayed a significant reduction of virulence, only causing increased body temperature of four pigs, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; inoculation with PRV JS-2012 strain into 7-day-old piglets could lead to death of 100% (5/5) of inoculated piglets, while PRV JS-2012-R strain displayed a significant reduction of virulence, only causing increased body temperature of four pigs, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy.

Through the pathogenicity test it indicated that compared with the parent virulent strain, i.e. PRV PRV-ZJ01 strain, PRV-ZJ01-R strain displayed a significant reduction of pathogenicity, and was an attenuated virus strain; compared with the parent virulent strain, i.e. PRV HeN1 strain, PRV HeN1-R strain displayed a significant reduction of pathogenicity, and was an attenuated virus strain; compared with the parent virulent strain, i.e. PRV JS-2012 strain, PRV JS-2012-R strain displayed a significant reduction of pathogenicity, and was an attenuated virus strain.

Meanwhile, in order to verify the stability of pathogenicity of different passages of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain, a group of piglets (5 piglets) at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with 1 ml (10 7.0 TCID 50 /ml) of the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV PRV-ZJ01-R strain by intranasal instillation, respectively, and another five piglets were used as the blank control group; a group of piglets (5 piglets) at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with 1 ml (10 7.0 TCID 50 /ml) of the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HeN1-R strain by intranasal instillation, respectively, and another five piglets were used as the blank control group; a group of piglets (5 piglets) at 7 days of age which were negative for pseudorabies antigens and antibodies were inoculated with 1 ml (10 7.0 TCID 50 /ml) of the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV JS-2012-R strain by intranasal instillation, respectively, and another five piglets were used as the control group. The clinical manifestations of piglets were observed and recorded daily until 28 days after inoculation of virus.

It showed in the results that, from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV PRV-ZJ01-R strain, PRV PRV-ZJ01-R strain displayed a significant reduction of virulence, only causing increased body temperature of 4˜5 pigs/group, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HeN1-R strain, PRV HeN1-R strain displayed a significant reduction of virulence, only causing increased body temperature of 4˜5 pigs/group, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy; from the observation of piglets for 28 days after inoculation with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV JS-2012-R strain, PRV JS-2012-R strain displayed a significant reduction of virulence, only causing increased body temperature of 4˜5 pigs/group, without any other clinical signs, or any changes of tissues or organs obtained from the necropsy.

Through the pathogenicity test of different passages, it indicated that different passages of PRV PRV-ZJ01-R strain, HeN1-R strain and PRV JS-2012-R strain all displayed lower virulence.

2. Immunogenicity Assay

On the 21 4 day after immunization, all the five piglets inoculated with PRV PRV-ZJ01-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV PRV-ZJ01 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 16.

On the 21 4 day after immunization, all the five piglets inoculated with PRV HeN1-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV HeN1 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 16.

On the 21 4 day after immunization, all the five piglets inoculated with PRV JS-2012-R strain and five piglets in the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV JS-2012 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 16.

The results indicated that all the piglets inoculated with PRV PRV-ZJ01-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with PRV HeN1-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with PRV JS-2012-R strain were healthy and alive, while all from the control group died.

According to the immunogenicity assay, the PRV PRV-ZJ01-R strain can provide excellent protection against PRV PRV-ZJ01 strain, showing excellent immunogenicity; the PRV HeN1-R strain can provide excellent protection against PRV HeN1 strain, showing excellent immunogenicity; the PRV JS-2012-R strain can provide excellent protection against PRV JS-2012 strain, showing excellent immunogenicity.

Meanwhile, in order to verify the stability of immunogenicity of different passages of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain, on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV PRV-ZJ01-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV PRV-ZJ01 strain; after challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed; on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HeN1-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV HeN1 strain; after challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed; on the 21 st day after immunization, all the immune groups inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV JS-2012-R strain as well as the control group were challenged with 1×10 7.0 TCID 50 /piglet of PRV JS-2012 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed.

The result indicated that all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV PRV-ZJ01-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV HeN1-R strain were healthy and alive, while all from the control group died; all the piglets inoculated with the cultures of the 1 st , 30 th , 60 th , 85 th and 110 th passages of PRV JS-2012-R strain were healthy and alive, while all from the control group died.

According to the immunogenicity assay of different passages, the culture of different passages of PRV PRV-ZJ01-R strain all can provide excellent protection against PRV PRV-ZJ01 strain, showing excellent immunogenicity; the culture of different passages of PRV HeN1-R strain all can provide excellent protection against PRV HeN1 strain, showing excellent immunogenicity; the culture of different passages of PRV JS-2012-R strain all can provide excellent protection against PRV JS-2012 strain, showing excellent immunogenicity.

3. Reversion of Virulence Test of the Virus

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 5 groups, each with 6 piglets. 6 piglets of Group 1 which were negative for pseudorabies antigens and antibodies were inoculated with 10 7.0 TCID 50 /piglet of the cultures of PRV PRV-ZJ01-R strain (the 1 st , 30 th , 60 th , 85 th , 110 th passages and the 1 st +30 th +60 th +85 th +110 th passages) respectively by intranasal instillation. On day 14, they were raised together with the 6 piglets of Group 2 which were negative for pseudorabies antigens and antibodies. 14 days later, the 6 piglets of Group 1 were drawn back, and again, the 6 piglets of Group 3, which were negative for pseudorabies antigens and antibodies, were raised together with the 6 piglets of Group 2, and so on. After 4 serial passages, all the drawn piglets were killed in order to observe if there were any pathological changes.

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 5 groups, each with 6 piglets. 6 piglets of Group 1 which were negative for pseudorabies antigens and antibodies were inoculated with 10 7.0 TCID 50 /piglet of the cultures of PRV HeN1-R strain (the 1 st , 30 th , 60 th , 85 th , 110 th passages and the 1 st +30 th +60 th +85 th +110 th passages) respectively by intranasal instillation. On day 14, they were raised together with the 6 piglets of Group 2 which were negative for pseudorabies antigens and antibodies. 14 days later, the 6 piglets of Group 1 were drawn back, and again, the 6 piglets of Group 3 which were negative for pseudorabies antigens and antibodies, were raised together with the 6 piglets of Group 2, and so on. After 4 serial passages, all the drawn piglets were killed in order to observe if there are any pathological changes.

30 piglets at 7 days of age which were negative for pseudorabies antigens and antibodies were randomly divided into 5 groups, each with 6 piglets. 6 piglets of Group 1 which were negative for pseudorabies antigens and antibodies were inoculated with 10 7.0 TCID 50 /piglet of the cultures of PRV JS-2012-R strain (the 1 st , 30 th , 60 th , 85 th , 110 th passages and the 1 st +30 th +60 th +85 th +110 th passages) respectively by intranasal instillation. On day 14, they were raised together with the 6 piglets of Group 2 which were negative for pseudorabies antigens and antibodies. 14 days later, the 6 piglets of Group 1 were drawn back, and again, the 6 piglets of Group 3, which were negative for pseudorabies antigens and antibodies, were raised together with the 6 piglets of Group 2, and so on. After 4 serial passages, all the drawn piglets were killed in order to observe if there are any pathological changes.

It showed in the results that no abnormal changes were found during the clinical observation and gross anatomy of 30 experimental piglets infected with PRV-ZJ01-R strain, 30 experimental piglets infected with HeN1-R strain, and 30 experimental piglets infected with JS-2012-R strain by the 4th serial passage of the cohabitation infection experiment, indicating that there was no reversion of virulence of the three attenuated strains. Therefore, the safety of the vaccines can be ensured since the viruses, after being inoculated into piglets, would not evolve into virulent viruses which are able to cause disease.

4. Genes Sequences Analysis

The genome amplification of the cultures of the 1 st passage to 110 th passage of PRV-ZJ01-R strain was accomplished by means of RT-PCR (The genomic DNA of the culture of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acid sequence of the virus. The obtained amino acids sequence was compared with the amino acids sequence of the parent virulent strain, i.e. PRV-ZJ01 strain via software for sequence analysis, and the amino acids sequence of the virus was characterized.

The genome amplification of the cultures of the 1 st passage to 110 th passage of PRV HeN1-R strain was accomplished by means of RT-PCR (The genomic DNA of the cultures of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acid sequence of the virus. The obtained amino acids sequence was compared with the amino acids sequence of the parent virulent strain, i.e. PRV HeN1 strain via software for sequence analysis, and the amino acids sequence of the virus was characterized.

The genome amplification of the cultures of the 1 st passage to 110 th passage of PRV JS-2012-R strain was accomplished by means of RT-PCR (The genomic DNA of the culture of different passages was amplified respectively). The product acquired from amplification was recovered, purified, and linked to the plasmid vector for sequencing, so that the nucleotide sequence of the viral gene was determined and transformed through computer software into the amino acids sequence of the virus. The obtained amino acids sequence was compared with the amino acids sequence of the parent virulent strain, i.e. PRV JS-2012 strain via software for sequence analysis, and the amino acids sequence of the virus was characterized.

It showed in the results that for the cultures of the 1 st passage to 110 th passage of PRV PRV-ZJ01-R strain, the amino acids encoded by each viral genes commonly have continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total, of which the deficiency site and size are totally the same as those of PRV HN1201-R strain; for the cultures of the 1 st passage to 110 th passage of PRV HeN1-R strain, the amino acids encoded by each viral genes commonly have continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total, of which the deficiency site and size are totally the same as those of PRV HN1201-R strain; for the cultures of the 1 st passage to 110 th passage of PRV JS-2012-R strain, the amino acids encoded by each viral genes commonly have continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total, of which the deficiency site and size are totally the same as those of PRV HN1201-R strain; compared with their parent virulent strains, each of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain has continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total.

It indicated that a common characteristic change of the amino acids encoded by the viral genes of the different passages of cultures of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain is consistent with that of the amino acids encoded by the viral genes of PRV HN1201-R strain, which is caused by the deficiency of genes encoding said amino acids sequences, showing the stability of the method of attenuating the PRV by passage according to the present invention, and in the meanwhile, further indicating that continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total, in PRV is the reason for the reduction of virulence of its parent virulent strain.

Example 14: Preparation of the Attenuated Live Vaccines of PRV PRV-ZJ01-R Strain, PRV HeN1-R Strain and PRV JS-2012-R Strain

The attenuated live vaccines of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain were prepared according to the procedure in Example 3. The specific ratios of contents of the vaccines are shown in Table 17.

Example 15: Immunogenicity Assay of the Attenuated Live Vaccines of PRV PRV-ZJ01-R Strain, PRV HeN1-R Strain and PRV JS-2012-R Strain

30 9-day-old piglets which were negative for PRV antigens and PRV antibodies were randomly divided into 6 groups, each with 5 piglets, and the piglets were immunized with the attenuated live vaccines of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain prepared in Example 3. Piglets in Group 1 were immunized with Vaccine 6, those in Group 3 were immunized with Vaccine 7 and those in Group 5 were immunized with Vaccine 8, and Groups 2, 4 and 6 were all the control groups. On day 21 after immunization, the piglets in Groups 1 and 2 were challenged with 1×10 7.0 TCID 50 /piglet of PRV PRV-ZJ01 strain, those in Groups 3 and 4 were challenged with 1×10 7.0 TCID 50 /piglet of PRV HeN1 strain, and those in Groups 5 and 6 were challenged with 1×10 7.0 TCID 50 /piglet of PRV JS-2012 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The results are shown in Table 18.

The results indicated that immunizing piglets with the attenuated live vaccines of PRV-ZJ01-R strain prepared in example 14 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge; immunizing piglets with the attenuated live vaccine of PRV HeN1-R strain prepared in example 14 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge; immunizing piglets with the attenuated live vaccine of PRV JS-2012-R strain prepared in example 14 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge.

It has proven that the attenuated live vaccines of PRV PRV-ZJ01-R strain, PRV HeN1-R strain and PRV JS-2012-R strain can provide excellent protection, showing excellent immune protection and safety; meanwhile it indicated again that continuous deficiency of gI/gE/11K/28K genes, 3455 bp in total, from the PRV virus would have no effect on its immunogenicity.

Example 16: Broad-Spectrum Immunogenicity Assay of the Attenuated Live Vaccine of PRV HN1201-R Strain

50 9-day-old piglets which were negative for PRV antigens and PRV antibodies were randomly divided into 10 groups, each with 5 piglets, and the piglets were immunized with the attenuated live vaccine of PRV HN1201-R strain prepared in Example 3. Piglets in Groups 1, 3, 5, 7 and 9 were immunized with Vaccine 1, and Groups 2, 4, 6, 8 and 10 were the control groups. On day 21 after immunization, the piglets in Groups 1 and 2 were challenged with 1×10 7.0 TCID 50 /piglet of PRV HN1202 strain, those in Groups 3 and 4 were challenged with 1×10 7.0 TCID 50 /piglet of PRV Fa strain, those in Groups 5 and 6 were challenged with 1×10 7.0 TCID 50 /piglet of PRV PRV-ZJ01 strain, those in Groups 7 and 8 were challenged with 1×10 7.0 TCID 50 /piglet of PRV HeN1 strain, and those in Groups 9 and 10 were challenged with 1×10 7.0 TCID 50 /piglet of PRV JS-2012 strain. After challenge, the body temperature of piglets was measured daily, and in the meanwhile clinical signs and death status were observed. The specific results are shown in Table 19.

The results indicated that immunizing piglets with the attenuated live vaccine of PRV HN1201-R strain prepared in Example 3 can block virus infection (i.e. prevent occurrence of clinical signs), and provide 100% (5/5) protection rate for piglets, while all the piglets in the blank control group died by day 5 after challenge.

It has proven that the attenuated live vaccine of PRV HN1201-R strain prepared according to the present invention can provide a fully protection against the epidemic PRV from different sources, showing excellent broad-spectrum immunogenicity.

Those are only preferred embodiments of the present invention as described above, which cannot be used to limit the present invention in any forms. Although the present invention has been revealed as described above in the form of the preferred embodiments, they are not intended to limit the present invention. Any skilled in the art can make several changes to the above technical content or modify the above technical content as equivalent embodiments with equivalent substitution, without departing from the technical scope of the present invention. Any simple change, equivalent substitution or modification etc, which are made to the above embodiments, based on the technical nature of the present invention, without departing from the content of technical solution of the present invention, should fall within the scope of protection of the present invention.