Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine

A technology for porcine circovirus disease and porcine circovirus, which is applied in the directions of viruses/bacteriophages, biochemical equipment and methods, antiviral agents, etc., can solve food safety problems, increase the probability of mixed infection, serious problems, etc., and achieve virulence Strong, reduce pig stress, high fecundity effect

Inactive Publication Date: 2013-11-27
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the only vaccines used on the market are inactivated vaccines for the prevention of Streptococcus suis, Haemophilus parasuis and porcine circular disease alone
The inactivated vaccines for simultaneously preventing streptococcosis suis, Haemophilus parasuis and porcine circus disease have not been reported at home and abroad
Moreover, domestic bacterial diseases are mainly prevented and controll

Method used

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  • Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine
  • Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine
  • Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Isolation, screening and identification of porcine circovirus type 2

[0040] 1. Virus Isolation and Culture

[0041] The diseased material to be isolated—the diseased material of each tissue was isolated in September 2004 by the inventor of the applicant in a large-scale pig farm in Hubei with naturally ill pigs. Suspected piglets with post-weaning multisystemic wasting syndrome (PMWS) were selected, and tissues such as lungs and lymph nodes were collected. The specific separation method is as follows: after the tissue disease material is washed with sterile physiological saline, an appropriate amount of DMEM medium is added to a grinder to grind into a tissue suspension, repeated freezing and thawing 3 times, centrifuging at 5000rpm / min for 25 minutes, and taking the supernatant , 0.22um microporous membrane sterilized, stored at -20°C or ready to use. Take part of the disease tissue to extract DNA, and use the PCR method to detect it. Take out the positi...

Embodiment 2

[0055] Example 2: Sequence Analysis of Porcine Circovirus Type 2-WH Strain

[0056] Sequence analysis and evolutionary analysis were carried out on the genome of the identified porcine circovirus type 2-WH strain, as follows:

[0057] (1) Primer design

[0058] Referring to the nucleotide sequence of the PCV2 strain (AY122275) published in GenBank, a pair of primers were designed to amplify the target fragment with a size of 1767 bp, and the overlapping sequences of the two amplified fragments were used to splice the full-length viral genome. The primer sequences are as follows:

[0059] P 3 : 5’-GCACCGCGGAAATTTCTGACAAACGTTACA-3’

[0060] P 4 : 5'-GAACCGCGGGCTGGCTGAACT TTTGAAAGT-3' primer was synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0061] After the nucleotide sequence was determined, it was analyzed with DNAStar6.0. Nucleotide and phylogenetic tree analysis were carried out with the following isolates from different places (see Table 1).

[0062] Table ...

Embodiment 3

[0067] Embodiment 3: the mensuration of porcine circovirus type 2-WH strain TCID50

[0068] The PK-15 cells positive for porcine circovirus type 2-WH strain of the 5th and 25th generations of blind passage were repeatedly frozen and thawed at -20°C for 3 times, and the PK-15 cells that grew well (without PCV contamination) were treated with 0.05% trypsin Digested, dispensed into 96-well plates, 90 μL per well, and then added DMEM medium containing 10% serum for 10 -1 -10 -12 Diluted virus fluid. Inoculate 6 wells for each dilution, mix well and put in CO 2 Continue to grow in the incubator. Discard culture fluid after 12h, process 30min with 300mmol / L D-glucosamine, add the DMEM culture fluid that contains 2% serum after PBS washing and continue to cultivate 48h; Discard culture fluid, do indirect immunofluorescence test ( IFA) judgment result. Calculate the TCID of virus strains by Reed-Muench method 50 .

[0069] The isolated porcine circovirus type 2-WH strain has a vi...

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Abstract

The invention discloses a trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, a preparation method of the vaccine and applications of the vaccine. Porcine circovirus 2-WH has an accession number of CCTCC NO: V20133 and has strong virulence, good immunogenicity, a high antigen titer, and a high virulence reaching 10<7.4>TCID[50]/mL. The trigeminy inactivated vaccine, which is prepared by mixing the porcine circovirus 2-WH, porcine streptococcus suis 2-LT, and porcine haemophilus parasuis 4-MD0322 and porcine haemophilus parasuis 5-SH0165, can simultaneously prevent the porcine circovirus disease and diseases caused by porcine streptococcus suis 2-type, porcine haemophilus parasuis 4-type and porcine haemophilus parasuis 5-type, the immune protective effect on the same serotype reaches over 80%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a triple inactivated vaccine for porcine circovirus disease, suis streptococcosis and haemophilus parasuis disease, and also relates to a porcine circovirus disease, suis streptococcosis and parasuis haemophilus disease The preparation method of the bacillosis triple inactivated vaccine also relates to the application of a triple inactivated vaccine of porcine circovirus disease, suis streptococcus disease and Haemophilus parasuis disease. The triple inactivated vaccine prepared by the invention is mainly suitable for sows, boars and piglets, and has good immune effect. technical background [0002] Porcine circovirus type 2 infection is the main cause of multisystemic wasting syndrome (PMWS) in weaned pigs, and other synergistic factors can enhance the severity of the disease. Porcine circovirus disease can cause the infected pigs to become thin, grow slowly, and reduce feed remune...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/295A61P31/20A61P31/04
Inventor 徐高原康超周明光严卫东陈波陈章表陈关平金梅林何启盖陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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