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Pig Getah virus strain, vaccine composition, and preparation method and application thereof

A vaccine composition and virus technology, applied in biochemical equipment and methods, microorganism-based methods, vaccines, etc., can solve the problems of strong virulence, lack of inactivated Getavirus vaccines, etc., to prevent epidemics and spread, The effect of reducing the incidence of piglets and reducing economic losses

Active Publication Date: 2017-05-10
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The outbreak and harm of the disease in Japanese horse racing in 2014 have aroused people's attention again, and the pathogenic mechanism and prevention and control measures of the disease need to be further studied
It can be seen from the above vaccines that most Geta vaccines are mainly used for horses, and they are mainly attenuated live vaccines, which have a certain risk of virulence reversion. Moreover, the strains used are at least 20 years ago, which are different from those in recent years. Compared with the detected strains, there are certain mutations, and they do not necessarily have good protection against the current circulating strains. Therefore, there is still a lack of a new safe and efficient inactivated Getavirus vaccine, especially for pigs. inactivated vaccine

Method used

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  • Pig Getah virus strain, vaccine composition, and preparation method and application thereof
  • Pig Getah virus strain, vaccine composition, and preparation method and application thereof
  • Pig Getah virus strain, vaccine composition, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Isolation, identification and biological characteristics of porcine getavirus HNJZ-S1 strain

[0032] 1.1 Experimental materials

[0033] The disease samples used in the experiment came from aborted fetus samples from a pig farm in Jiaozuo, Henan Province, collected in January 2016; the Marc-145 monoclonal cell line was frozen and stored in our laboratory; male and female Kunming mice were purchased from the Experimental Animal Center of Henan Province.

[0034] 1.2 Primer design

[0035] Referring to the GETV HB0234 (EU015062) gene sequence published in GenBank, a pair of specific primers were designed using Premier5, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The nucleotide sequence of the specific primer is as follows: PF: 5'-AACCCACCCGTGCGCTTGT-3' (SEQ ID NO.1); PR: 5'-CGCCGTTGTTGTCTGATTGAGG-3' (SEQ ID NO.2).

[0036] 1.3 RT-PCR detection of GETV RNA in tissue disease materials

[0037] Collect the viscera of pig abo...

Embodiment 2

[0053] Example 2 Cloning and sequence analysis of the complete genome of porcine getavirus HNJZ-S1 strain

[0054] 2.1 Primer design

[0055] Referring to the GETV HB0234 (EU015062) gene sequence published in GenBank, Premier5 was used to design 12 pairs of specific primers (see Table 2 for its nucleotide sequence); and 3 primers were designed for the 5'UTR according to the RACE kit instructions (these The nucleotide sequence is shown in Table 3), including 1 reverse transcription primer, 1 inner primer and 1 outer primer; 1 inner primer and 1 outer primer were designed for the 3'UTR (the nucleotide sequence is shown in Table 3), primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0056] 2.2 RNA extraction and whole genome amplification of porcine getavirus HNJZ-S1 strain

[0057] The harvested SGETV cell culture was repeatedly frozen and thawed 3 times to lyse the cells, centrifuged at 8000r / min for 5min, and the supernatant was taken. The total RNA of the disea...

Embodiment 3

[0073] The preparation of embodiment 3 porcine getavirus inactivated vaccine

[0074] 3.1 Virus culture

[0075] Use DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 g / mL streptomycin at pH 7.2 at 37°C in 5% CO 2 Culture the Marc-145 cells adherently in the incubator; discard the culture medium when the cell growth coverage rate reaches 80%-90%, and inoculate the 4th generation cell passage of porcine getavirus HNJZ-S1 strain according to the multiplicity of infection of 0.1, and then use DMEM medium containing 2% fetal bovine serum, 100 U / mL penicillin, 100 g / mL streptomycin, 37°C, 5% CO 2 Cultivate in an incubator, cultivate for 48h-60h, and harvest the virus liquid after the cytopathy is complete, freeze and thaw three times at -80°C and room temperature, centrifuge at 10,000×g for 10 minutes, remove the cell debris, and obtain the supernatant to obtain the porcine getavirus liquid. Store frozen at -80°C.

[0076] 3.2 Inactivation of virus liqu...

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Abstract

The invention discloses a newly separated pig Getah virus HNJZ-S1 strain. The microbial preservation number of the pig Getah virus HNJZ-S1 strain is CGMCC NO 12550; the pig Getah virus is separated from a naturally infected pig group for the first time in China, and has good immunogenicity. The invention also discloses a vaccine composition which is prepared from the pig Getah virus HNJZ-S1 strain, and a preparation method thereof. The vaccine composition contains inactivated pig Getah virus strain HNJZ-S1 strain and a pharmaceutically acceptable adjuvant; the preparation method for the vaccine composition comprises the steps of virus culturing, virus liquid inactivating, aqueous phase preparing, oil phase preparing emulsifying and the like. The vaccine composition has good immunogenicity, and can produce high immunity after immunization; the attacking protecting rate is high; after inoculation, the abortion rate and the stillbirth rate of sows are obviously reduced; the incidence of piglets is remarkably reduced; the popularizing and spreading of the pig Getah virus can be effectively prevented; the vaccine composition has a broad application prospect.

Description

technical field [0001] The invention relates to a porcine gaita virus strain, and also relates to an inactivated vaccine composition prepared from the porcine gaita virus strain and a preparation method thereof, belonging to the field of isolation and application of porcine gaita virus strains. Background technique [0002] Getah virus (GETV) is an arbovirus with mosquitoes as the main transmission medium. It is mainly asymptomatic or high-titer viremia in natural hosts, but it is transmitted to susceptible animals through mosquitoes. Can cause viral disease epidemic. The virus was first isolated from Culex snowback in Malaysia in 1955. Since then, in Japan, the eastern part of the former Soviet Union, Southeast Asia and Australia, Gehavirus has also been isolated from the blood of Culex tribandi, Aedes stinging mosquitoes and pigs. Since the disease has been paid attention to, the presence of Getavirus antibodies has been detected in pigs, horses, cattle, goats, dogs, rab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252A61K2039/552C12N7/00C12N2770/36121C12N2770/36134
Inventor 王川庆常洪涛崔丹丹周峰赵军陈陆杨霞王新卫王新港王傲杰李永涛刘红英姚惠霞
Owner HENAN AGRICULTURAL UNIVERSITY
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