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Low-pathogenicity porcine reproductive and respiratory syndrome virus, and vaccine and application thereof

A respiratory syndrome, low pathogenicity technology, applied in the direction of antiviral agents, virus/bacteriophage, virus antigen components, etc., can solve the problem of decreased protective effect of vaccines and achieve a good immune response effect

Active Publication Date: 2016-07-20
兆丰华生物科技(南京)有限公司北京生物医药科技中心 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing vaccines show better protective effects against strains with higher homology, but as the homology with vaccine strains decreases, the protective effect of vaccines also decreases

Method used

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  • Low-pathogenicity porcine reproductive and respiratory syndrome virus, and vaccine and application thereof
  • Low-pathogenicity porcine reproductive and respiratory syndrome virus, and vaccine and application thereof
  • Low-pathogenicity porcine reproductive and respiratory syndrome virus, and vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The isolation and identification of embodiment 1PRRS virus

[0025] 1. Isolation of porcine reproductive and respiratory syndrome virus

[0026] The pig lung samples sent by the Hebei pig farm for inspection were ground and tested by RT-PCR method, and the result was positive for porcine reproductive and respiratory syndrome virus.

[0027] Take an appropriate amount of the lungs for inspection and grind them. The grinding solution is PBS and added in a ratio of 1:5 (weight: volume). The ground product was repeatedly frozen and thawed three times, centrifuged at 12000r / min for 10min at low temperature, and the supernatant was taken and filtered through a 0.22μm filter membrane.

[0028] Inoculate overgrown Marc-145 cells according to the conventional method (wash three times with PBS of pH 7.4 before inoculation), inoculate the virus at a ratio of 10%, absorb at 37°C for 30 minutes, supplement the cell maintenance solution, and incubate at 37°C in an incubator. Observ...

Embodiment 2

[0037] The plaque purification of embodiment 2PRRS virus

[0038] Take the 4th generation virus of PRRSVHB2014001 strain and carry out 10-fold ratio dilution with 2% DMEM culture medium, and the dilution ratio is 10 -1 to 10 -8 , take 10 -3 to 10 -8 Add 500 μl of the dilution solution to a 6-well cell plate full of MARC-145 cells, incubate in a 37°C incubator for 1 hour, discard the virus solution, wash once with PBS, and add 4ml of 1% low-melting point agarose to cover the cells. After the agarose was cooled, put it into the cell culture incubator and culture it at 37°C for 3-5 days. When obvious plaques were formed, pick the monoclonal plaques and inoculate them into the overgrown monolayer of MARC-145 cells for expansion. The amplified virus was again subjected to two rounds of plaque purification as described above. After three rounds of plaque purification, the HB2014001 strain was expanded and cultured with MARC-145 cells, and stored at -80°C after aliquoting.

Embodiment 3

[0039] The animal pathogenicity test of embodiment 3PRRSVHB2014001 strain

[0040] 1. Animal pathogenicity test of PRRSVHB2014001 strain

[0041] Six 4-week-old piglets with double-negative PRRSV antigen and antibody were screened, and they were randomly divided into two groups. One group injected 1ml into the back of the neck with a total of 10 piglets. 5 TCID 50 Virus solution, another group of piglets were injected with 1ml of 2% MDEM culture solution at the same site as a control.

[0042] After challenge, the experimental pigs were checked for clinical symptoms and rectal body temperature every day. The results of body temperature monitoring showed that before and after the challenge, the body temperature of the piglets in the control group fluctuated within the normal range; the body temperature of the piglets began to rise slightly on the second day after inoculation with the PRRSVHB2014001 strain, and the body temperature all rose to about 40°C, with a maximum of 40....

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Abstract

The invention relates to a low-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV), and a vaccine and an application thereof. The PRRSV is separated from porcine lung tissues by using Marc-145 cells, the above separated strain can proliferate on the Marc-145 cells and make the Marc-145 cells have typical cytopathy, the Marc-145 is detected to obtain the tilter of the separated strain of 10<7.0> TCID50 / mL, and the strain is preserved in China General Microbiological Culture Collection Center on Oct. 22, 2015 with the preservation number of CGMCC No.9819; and leaned piglets artificially inoculated with the new virus strain only have weak thermal response, have no obvious clinic symptoms, and normally grow without untoward effects. The new strain artificially inoculated to pigs can induce the pigs to have good immune response I order to generate high level PRRSV antibodies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of low pathogenic porcine reproductive and respiratory syndrome virus and its application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is one of the pig diseases that cause serious economic losses in pig-raising countries. Hopper, et al., 1992; Yang Hanchun et al., 2006). The primary pathogen of the disease is porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, RRSV), equine arteritis virus (Equinearteritis virus, EAV), mouse lactate dehydrogenase increased disease virus (Lactatedehydrogenase-elevating virus, LDV) and monkey Hemorrhagic fever virus (Simianhemorrhagicfevervirus, SHFV) belongs to the nest virus order, Arteriviridae, Arterivirus (Dea, et al., 2000; Snijder, et al., 1998). [0003] The disease first occurred in...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/12A61P31/14C12R1/93
Inventor 张家龙张国庆刘淑清徐冉王贵华赵亚荣
Owner 兆丰华生物科技(南京)有限公司北京生物医药科技中心
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