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Gene recombined swine cholera salmonella choleraesuis vaccine for blue-ear disease and application thereof

A technology for salmonella and porcine PRRS virus, which is applied in the direction of bacteria, antibacterial drugs, antiviral agents, etc., can solve the problems of harmful resistance to organisms and easy loss of plasmids, and achieve good biological safety and good protection-free Effect

Active Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two studies have a common feature, that is, all the plasmids used are resistance plasmids, which are easily lost in a non-resistant environment, and resistance is also harmful to the body.

Method used

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  • Gene recombined swine cholera salmonella choleraesuis vaccine for blue-ear disease and application thereof
  • Gene recombined swine cholera salmonella choleraesuis vaccine for blue-ear disease and application thereof
  • Gene recombined swine cholera salmonella choleraesuis vaccine for blue-ear disease and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Example 1 Preparation Example 1 (Preparation of gene fragments capable of prokaryotic expression of highly pathogenic PRRSV GP5m protein)

[0059] 1. Design and synthesis of primers

[0060] Refer to the reported PRRSV WUH3 (document: Li B, 2009; PRRSV WUH3 genome sequence see Genebank accession number: NO.HM853673) to design 2 pairs of primers (see Table 1), respectively amplify the genes with EcoR Ⅰ+BamH Ⅰ The A epitope of the restriction site, a 39bp PADRE is introduced into this downstream primer (ORF5m-AR); the B epitope of the BamH Ⅰ+HindⅢ restriction site), the primers required in the present invention are shown in Table 1.

[0061] 2. RNA extraction, reverse transcription and gene amplification

[0062] (1) Extraction of RNA

[0063]Use the RNA extraction kit purchased from Bioflux (operate according to the kit instructions) to extract the RNA of PRRSVWUH3, the specific method is as follows:

[0064] 1) Take 100 μL of PRRSV virus stock solution and add it to ...

Embodiment 2

[0088] Example 2 Preparation Example 2 (Construction and Identification of Recombinant Plasmid pYA-GP5m)

[0089] The first step in this example is to recover the A epitope product with EcoR Ⅰ+BamH Ⅰ restriction site, and use EcoR Ⅰ and BamH Ⅰ to recover the product of the A epitope and the balanced expression plasmid pYA3493 (see Figure 4 , the non-resistant prokaryotic expression plasmid pYA3493 (asd + , p-lactamasesignal sequence, pBRori) was transformed into the host strain Escherichia coli x6097 (the basic structure of the strain is araΔ(lac-pro)rpslΔasdA4Δ[zhf-2::Tn10]thiФ80d / lacZΔM15)), and double digestion, recovery and ligation The recombinant plasmid was constructed and named pYA-A, the ligated product was transformed into Escherichia coli x6097, positive clones were screened on a DAP negative plate, and the pYA-A plasmid was extracted. The second step is to use BamHI and HindIII to digest, recover and connect the B epitope recovery product with BamH Ⅰ + HindIII re...

Embodiment 3

[0139] Example 3 Preparation Example 3 (Construction and identification of recombinant strain C501-GP5m expressing GP5m protein)

[0140] The recombinant plasmid pYA-GP5m was extracted from Escherichia coli x6097 lacking the asd gene with a plasmid mini-extraction kit (purchased from Transgene Company), and then electrotransformed into △asdC501 competent cells, positive clones were screened on DAP negative plates, and picked A single colony was cultured, PCR identification was carried out with identification primers pYA-F / pYA-R, and sequence determination was carried out. The results showed that the obtained recombinant Salmonella choleraesuis containing the pYA-GP5m plasmid was correct, and the applicant named the recombinant strain For Salmonella choleraesuis C501-GP5m, its construction process is as follows figure 1 shown. Carry out PCR identification to this Salmonella choleraesuis C501-GP5m respectively with identification primer pYA-F / pYA-R based on plasmid pYA design, ...

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Abstract

The invention relates to a gene recombined swine cholera salmonella choleraesuis vaccine for a blue-ear disease and application thereof, belongs to the technical field of animal bacteria genetic engineering, and particularly relates to construction of recombined swine cholera salmonella choloraesuis without resistance maker and used for expressing main immunogenicity membrane protein of porcine reproductive and respiratory syndrome virus(PRRSV) and preparation and application of a live vaccine. Recombined swine cholera salmonella choleraesuis C501-GP5m expressing the swine PRRSV GP5m protein is gained and preserved in the China model culture preservation centre, and the preservation number is CCTCC NO: M2012275. The invention further discloses a method for preparing swine cholera salmonella choleraesuis and the porcine reproductive and respiratory syndrome virus vaccine from the recombination strain and application thereof. The prepared recombined live vaccine can stimulate swine to develop the immunity to resist swine cholera salmonella choloraesuis and PRRSV main immunogenicity membrane protein GP5m, and can effectively prevent the infection of paratyphus suum and PRRSV.

Description

technical field [0001] The invention belongs to the technical field of animal bacterial genetic engineering vaccines, in particular to the construction and application of a recombinant Salmonella choleraesuis expressing a modified ORF5m gene of highly pathogenic porcine reproductive and respiratory syndrome virus (or porcine blue ear disease) and a vaccine . Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), which can cause severe reproductive disorders in pregnant sows and respiratory symptoms in piglets. viral infectious disease (Meulenberg et al, 2000), also known as PRRS. The disease is characterized by reproductive disorders such as premature birth, abortion, stillbirth, mummified fetus, and weak piglets in pregnant sows, high mortality of newborn and pre-weaned pigl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/295A61K39/12A61K39/112A61P31/14A61P31/04C12R1/42
Inventor 何启盖杨昆郭爱珍陈焕春刘晓莉
Owner HUAZHONG AGRI UNIV
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