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Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof

A Salmonella typhi and vector vaccine technology, applied in the field of bioengineering, can solve the problems of low total antibody level, difficulty in large-scale application, and low expression level, and achieve the effect of avoiding cumbersome processes and defects, easy acceptance, and high antibody level

Inactive Publication Date: 2010-12-22
NORTHWEST A & F UNIV
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  • Claims
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Problems solved by technology

However, the eukaryotic expression vector system has defects such as complicated operations and low expression levels, making it difficult to apply on a large scale
In fact, the bigger problem with the above-mentioned genetic vaccines is that the antibody level produced by the first immunization is relatively low, and two immunizations are required, but even if the two immunizations are performed, the total antibody level is still lower than that of ordinary inactivated vaccines.
In addition, according to the above-mentioned report "Oral Immune Response of Porcine Reproductive and Respiratory Syndrome Virus_PRRSV_DNA Vaccine Mediated by Attenuated Salmonella", it is still difficult to determine whether oral PRRSV DNA vaccine can induce mucosal immunity

Method used

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  • Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof
  • Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof
  • Recombinant attenuation salmonella typhimurium vector vaccine expressing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the cloning of ORF5 gene of PRRSV Shaanxi strain

[0047] 1.1 Design of primers

[0048] According to the published genome sequence of PRRSV CH-1a strain (GenBank: AY032626), a pair of specific primers (P1 / P2) was designed. Upstream primer P1: 5′-CCG GAATTCG C AGCAAC AAC AGC AGC TCTCA-3'5' end plus EcoR I restriction site, downstream primer P2: 5'-ACG GTC GAC CTA GAG ACG ACC CCA TTGTT-3', plus a SalI restriction site at the 5' end. The expected amplified fragment is 507bp, containing the complete ORF5 gene without the signal peptide gene.

[0049] 1.2 Extraction of viral genome

[0050] The cytotoxicity of the PRRSV SX strain (Shaanxi isolate) stored at -80°C was taken out, and viral RNA was extracted according to the Trizol extraction kit. Take 3 μL of RNA as template, add 1 μL of ORF5 downstream primer P2, 4 μL of dNTP, 0.5 μL of RNase-Inhibitor, 0.5 μL of AMV, 4 μL of 5×AMV Buffer, add DEPC H2O to 20 μL, and 42°C for 1 hour to obtain the cDNA te...

Embodiment 2

[0105] Embodiment 2, SDS-PAGE detects the expression of PRRSV GP5 protein

[0106] Recombinant bacteria X4550 (pYA3341-ORF5) and empty plasmid bacteria X4550 (pYA3341) were respectively inoculated in LB liquid medium containing 20 μg / ml NA and shaken at 37°C for 18 hours, then the supernatant was discarded by centrifugation, the bacteria were collected, and 0.01M ( After resuspending the cells in PBS solution (pH 7.2), add 5× protein loading buffer, boil for 15 minutes, collect the supernatant by centrifugation, and perform SDS-PAGE electrophoresis on a 12% gel. Prepare SDS-PAGE polyacrylamide gel. After the gel is completely polymerized, pull out the sample comb, wash the sample hole with negative buffer and dry the water in the hole with filter paper. The sample to be tested and the protein molecular weight standard are loaded at the same time, and the electrophoresis is stopped when the Coomassie brilliant blue reaches the bottom of the gel. The gel was fixed in the fixati...

Embodiment 3

[0109] Embodiment 3, Western blotting detects the immunoreactivity of GP5 protein

[0110] SDS-PAGE electrophoresis was performed according to the above method, and after the protein was separated by SDS-PAGE, it was electrotransferred to PVDF membrane. 5% skimmed milk powder or 3% bovine serum albumin buffer (10mmol / L Tris Cl, pH7.5, 150mmol / LNaCl, 0.05% Tween20) for blocking at 37°C for 2h, washing buffer (10mmol / L Tris Cl pH7. 5. Wash 3 times with 150mmol / L NaCl, 0.05% Tween20), 10-15min each time, dilute porcine anti-PRRSV positive serum with blocking buffer (1:300) to cover the membrane, feel at room temperature for 2h, wash 3-5 The second time, goat anti-pig IgG (1:2000) labeled with horseradish peroxidase was sensed at room temperature for 2 hours, washed 3 to 5 times, each time for 10 to 15 minutes, and finally washed once with distilled water, and the PVDF membrane was clipped out and dried slightly , prepare ECL (enhanced chemiluminescence) working solution, incubat...

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Abstract

The invention discloses a recombinant attenuation salmonella typhimurium vector vaccine expressing a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) immunogen gene and a preparation method and application thereof, and in particular relates to an oral attenuation salmonella typhimurium. The recombinant attenuation salmonella typhimurium has a sequence shown in SEQ ID NO.1, and can express PRRSV cyst membrane protein GP5. The attenuation salmonella typhimurium is inoculated in an LB (Lysogeny Broth) culture medium and cultured for 18h, and the bacterium concentration is regulated to be 1010CFU / ml, therefore, the safe and effective oral attenuation salmonella typhimurium live vector vaccine is prepared and used for preventing PRRS.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant attenuated Salmonella typhimurium vector vaccine expressing PRRSV immunogen gene and a preparation method thereof. Background technique [0002] PRRS (Porcine Reproductive and Respiratory Syndrome) is one of the severe infectious diseases of pigs caused by PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). It is listed as a B-type animal infectious disease by the International Veterinary Bureau, mainly causing reproductive disorders in sows (such as abortion, premature birth, stillbirth, mummified fetus) and respiratory symptoms in piglets. [0003] There are currently two serotypes of PRRS occurring in the world: the European type and the American type. So far only the American type is popular in our country. Because PRRS mainly causes sow reproductive barriers, it has caused huge economic losses to my country's pig industry. PRRSV belongs to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/39C12N15/41C12N15/74C12N1/21A61P31/14
Inventor 童德文许信刚张琪
Owner NORTHWEST A & F UNIV
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