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Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof

A technology of Bordetella blood and Salmonella, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unaccepted by people, biosafety issues, etc., and achieve good immune protection, good biosafety, and broad Effect of Market Application Prospects

Inactive Publication Date: 2008-04-09
WUHAN KEQIAN BIOLOGY CO LTD
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AI Technical Summary

Benefits of technology

This patented technology allows researchers to study how bacteria called Brachytherapy Tube works when they infect animals or humans. These treatments use special DNA that helps them fight off germs like salmons but also help it get ridged out quickly without causing any side reactions during treatment. They found that this new type of virus was more effective compared to older versions of these drugs while still being safe even if given multiple dosages over several months after administration. Overall, their technical results show promise for developing improved methods for protecting individuals who may be exposed to such diseases through vaccination strategies targeted specifically specific genotypes associated with certain disease types.

Problems solved by technology

This patented technical issue addressed in this patents relates to developing safe, effective and cost-effectively novel veterinary vims vaccinations targeted towards salmons caused by Brucella sppagiae disease virus(BVDV), which currently use only one type of killed vaccination called tissue culture infected cells/recompressed viruses. However, traditional approaches like recombined versions of these viratory pathogens could pose safety concerns if administered without proper handling procedures during manufacturing processes.

Method used

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  • Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof
  • Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof
  • Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of Salmonella choleraesuis C500asd gene deletion strain

[0047] 1. Primer design (for gene cloning and molecular detection)

[0048] Referring to the reported asd gene sequence of Salmonella typhimurium LT2 strain (GenBank No: AE008863), 2 pairs of primers (pa1 / pa2 and pa3 / pa4, see Table 1) were designed, from the Salmonella choleraesuis attenuated vaccine strain C500 (purchased from China Veterinary Medicine The upstream and downstream fragments asd1 (upper arm) and asd2 (lower arm) of the asd gene were respectively amplified in the genome of the commercial strain of the National Veterinary Microorganism Collection Center of the Inspection Institute. BamH I restriction site, BamH I and Kpn I restriction sites were introduced at the two ends of the lower arm respectively. In addition, 2 pairs of primers (pa5 / pa6 and pa7 / pa6, see Table 1) were designed to identify the C500 parental strain and asd-deleted strain. Primers were synthesized by Shang...

Embodiment 2

[0059] Example 2 Construction of recombinant plasmid pYA-F1P2

[0060] 1. Primer design

[0061] Two pairs of primers were designed according to the reported gene sequences of Bordetella bronchiseptica (Bordetella bronchiseptica) HH0809 (the strain Bordetella bronchiseptica) The F1 fragment of the fhaB gene and the P2 fragment of the prn gene were amplified in the genome preserved in the China Center for Type Culture Collection (CCTCC) on September 18, 2007 (the deposit number is CCTCC NO: M207148). The sizes of the amplified fragments were 465bp and 300bp, pf1 and pf2 were respectively introduced into EcoR I and Sal I restriction sites, and pp1 and pp2 were respectively introduced into SalI and HindIII restriction sites. The primer sequences are as follows:

[0062] pf1: 5'TTTAA GAATTC CTGACTGCCCTGGACAAT-3' (EcoRI)

[0063] pf2: 5'TTTAA GTC GAC TCGCAGATCCGCGGCAAA-3'(SalI)F1 fragment 465bp

[0064] pp1: 5'TAATT GTC GAC AACACCATGCTGCTGGTG-3'(SalI)

[0065] pp2: 5'TTT...

Embodiment 3

[0072] Example 3 Construction of recombinant strains expressing F1 and P2 gene fragments

[0073] The asd gene-deleted strain C501 of C500 loses the ability to synthesize DAP due to the deletion of the asd gene, so it cannot grow on a medium without exogenous DAP, but when it obtains the recombinant plasmid pYA-F1P2 containing the asd gene, it will obtain the ability to synthesize DAP ability to restore the ability to grow on DAP-free media. The asd gene deletion strain C501 of C500 was electrotransformed with the recombinant shuttle plasmid pYA-F1P2, and the positive clones were screened on the DAP negative plate, and a single colony was picked and cultured, and PCR identification was carried out with primers pa7 / pa6, pf1 / pf2 and pp1 / pp2 (See Figures 8A, 8B and 8C, respectively). The results showed that it was correct to obtain the C501 recombinant strain containing the pYA-F1P2 plasmid, and we named it C501(pYA-F1P2).

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Abstract

The invention pertains to the animal bacteria gene engineering field, relating to construction, vaccine preparation and application of salmonella choleraesuis of recombinant porcine with fhaB and prn gene fragment of porcine bordetella bronchiseptica from expression which contains no resistance markers. A strain of salmonella choleraesuis of recombinant porcine of C501 (pYA-F1P2 (preservation number is: CCTCC NO: M207138) with fhaB and prn gene fragment of porcine bordetella bronchiseptica from expression which contains no resistance markers is provided by the invention. The stain of asd gene lacks of samonella choleraesuis, and contains plasmid of pYA-F1P2 which can express asd in the strain and fhaB and prn gene fragment of porcine bordetella bronchiseptica. The invention discloses a preparation method of the recombination stain and vaccine and application in the preparation of the vaccine of the porcine salmonella choleraesuis and the porcine bordetella bronchiseptica. The gene engineering vaccine of the invention can stimulate porcine to produce an immune reaction to protect porcine from the salmonella choleraesuis and the porcine bordetella bronchiseptica and can effectively prevent from being infected by the salmonella choleraesuis and the porcine bordetella bronchiseptica.

Description

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Claims

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Application Information

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Owner WUHAN KEQIAN BIOLOGY CO LTD
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