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Recombinant attenuated salmonella typhimurium vector vaccine expressing PCV-2 immunogenic gene and preparation method thereof

A technology of Salmonella typhi and PCV-2, applied in the field of bioengineering, can solve the problem of no vaccine yet, achieve rapid antibody production, avoid cumbersome processes and defects, and be easily accepted

Inactive Publication Date: 2011-01-26
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no vaccine for the control of PCV-2

Method used

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  • Recombinant attenuated salmonella typhimurium vector vaccine expressing PCV-2 immunogenic gene and preparation method thereof
  • Recombinant attenuated salmonella typhimurium vector vaccine expressing PCV-2 immunogenic gene and preparation method thereof
  • Recombinant attenuated salmonella typhimurium vector vaccine expressing PCV-2 immunogenic gene and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0046] 1.1 Design of primers

[0047] According to the published genome sequence of PCV-2GX strain (GenBank: EF675237), a pair of specific primers (P1 / P2) was designed. Upstream primer P1: 5′-CCG GAATTCGCATGaatggcatcttc-3′ with an EcoR I restriction site at the 5′ end, and downstream primer P2: 5′-ACGGTCGAC AAGTGGGGGGTCTTTAAG-3′, with a Sal I restriction site at the 5′ end. The expected amplified fragment is 570bp, containing the complete ORF2 gene with the N-terminal nuclear localization region gene removed. 1.2 Extraction of viral genome

[0048] 1.2 Extraction of viral genome

[0049] The PCV-2 cytotoxicity stored at -80°C was taken out, and the viral DNA was extracted according to the instructions of the viral DNA extraction kit of Bao Biological Engineering (Dalian) Co., Ltd. The PCR reaction system is as follows: 5 μL of 10×PCRBuffer, 4 μL of dNTPs, 1 μL of P1 / P2, 1 μL of rTaq, 3 μL of viral DNA, and add water to 50 μL. Reaction program: denaturation at 95°C for 5min...

Embodiment 2

[0104] Recombinant bacteria X4550 (pYA3341-ORF2) 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 fixative solution for 30-60 minutes, heated to 60°C for Coomassie brilliant blue st...

Embodiment 3

[0107] 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% Tween (20), 10-15min each time, dilute porcine anti-PCV-2 positive serum with blocking buffer (1:300) to cover the membrane, feel at room temperature for 2h, wash for 3 ~5 times, horseradish peroxidase-labeled goat anti-pig IgG (1:2000) for 2h at room temperature, washed 3~5 times, each time for 10~15min, and finally washed once with distilled water, clamped out PVDF membrane slightly After drying, prepare ECL (enhanced chemiluminescence) working solution, incubate the membrane at room temperature under visible light for several minutes, wrap the imprinted membrane with plastic wrap and stic...

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Abstract

The invention discloses an oral recombinant attenuated salmonella typhimurium live vector vaccine as well as a preparation method and application thereof, particularly relating to an oral attenuated salmonella typhimurium. The recombinant attenuated salmonella typhimurium has a sequence shown in SEQ ID NO.1, and can express PCV-2 capsid protein ORF2. The attenuated salmonella typhimurium is inoculated in an LB culture medium for culturing for 18 hours; and the concentration of the attenuated salmonella typhimurium is regulated to 1010CFU / ml to prepare a safe and effective oral attenuated salmonella typhimurium live vector vaccine used for preventing porcine circovirus diseases.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant attenuated Salmonella typhimurium vector vaccine expressing PCV-2 immunogen gene and a preparation method thereof. Background technique [0002] Porcine circovirus (PCV) belongs to the genus Circovirus of the family Circoviridae and is a representative species of the genus Circovirus. DNA virus, the average particle diameter of which is 17nm, is the smallest animal virus found in veterinary medicine. [0003] PCV can be divided into two serotypes, PCV-1 and PCV-2. PCV-1 is non-pathogenic to pigs and is a non-pathogenic PCV. PCV-2 is pathogenic to pigs and can cause PMWS (multisystemic wasting syndrome in weaned piglets), also known as PMWS-related PCV. The disease broke out in Canada for the first time in 1991, and there were reports of the disease in many countries and regions in the world. At present, the disease exists in many pig farms in my country, ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/39C12N1/21C12N15/31A61P31/20
Inventor 童德文许信刚张琪
Owner NORTHWEST A & F UNIV
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