Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application

A technology for Salmonella and cholera suis, which is applied in the field of construction of recombinant Salmonella cholera suis live vaccine strains, can solve the problems of biosafety, unacceptable, large economic losses, etc., and achieves good biosafety and good immune protection. Effect

Inactive Publication Date: 2010-11-10
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The mortality rate of piglets over 5 weeks old is low, and adult pigs hardly die, but it often results in reduced production performance, lower feed remuneration, and greater economic losses (Sirinarumitr et al, 1998; Jones et al, 1997)
As in the past, expression plasmids carrying resistance genes were commonly used, but they were not accepted by people due to biosafety issues.

Method used

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  • Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application
  • Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application
  • Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Synthesis of Gene Fragment Containing Porcine Transmissible Gastroenteritis Virus Main Antigen Site

[0049] 1. Selection of main antigenic sites

[0050] The S protein of porcine transmissible gastroenteritis virus contains four antigenic sites, A, B, C, and D, among which the A and D antigenic sites play an important role in inducing neutralizing antibodies, and the A antigenic site is a major B cell antigen epitopes. Porcine transmissible gastroenteritis virus contains 4 main T cell epitopes, of which the N on the N protein 321 (321-335 residue region) cell antigen site can be the strongest T cell response, and can cooperate with B cell epitopes of different proteins in TGEV.

[0051] 2. Design of S and N gene primers and comparison of their homology

[0052] Two pairs of primers were designed with reference to the reported gene sequences of porcine transmissible gastroenteritis virus S and N (GenBank No: DQ443743), and S gene and N gene. The size of t...

Embodiment 2

[0061] Construction of embodiment 2 recombinant plasmid pYA-2SLN

[0062] 1. Cloning of SLN gene

[0063] The recombinant bacteria (DH5α / pET-2SLN) containing the SLN gene sequence were transferred to a bacterial bottle containing 3 mL of LB medium at a volume ratio of 1:100, and cultured overnight at 37°C and 200 rpm / min for 8 hours. According to the instructions of the bacterial plasmid extraction kit (purchased from Beijing BioFlux Company), the plasmid was extracted as a template for restriction digestion.

[0064] The SLN and pET-28a vectors (purchased from Treasure Bioengineering (Dalian) Co., Ltd.) were digested with EcoRI and NotI, recovered and purified, then ligated with T4DNA ligase, and then transformed into DH5α competent bacteria in a water bath at 16°C for 12h, and cultured at 37°C for 12h. Pick the bacteria into the liquid LB medium, shake and culture at 37°C and 200rpm / min for 12h, prepare a small amount of plasmids to obtain the plasmid pET-SLN (see Figure ...

Embodiment 3

[0076] Example 3 Construction of the Salmonella choleraesuis C500 / pYA-2SLN recombinant strain expressing the SLN gene fragment

[0077] The recombinant shuttle plasmid pYA-2SLN (see Example 2 for the source) was electrotransformed (parameters: voltage 2.0KV, time 4ms, capacitance 25μF and pulse resistance 200 ohms) to the C500ΔcrpΔasd deletion strain of Salmonella choleraesuis, and positive screening was performed on the DAP negative plate Cloning, picking a single colony for culture, and using primers pYASLNI / pYASLNII for PCR identification (PCR reaction system (25μL): 10×Buffer 2.5μL, 25mmol / L MgCl 2 2 μL, 2 μmol / L dNTPs 1 μL, 10 μg / mL upstream and downstream primers 1 μL each, 2U / μL Taq DNA polymerase 0.5 μL, sterile water 16 μL, template 1 μL. PCR reaction conditions: denaturation at 94°C for 4 minutes; then 25 cycles of 30s at 94°C, 30s at 56°C, and 60s at 72°C; and extension at 72°C for 10 minutes. PCR products were separated by 80V electrophoresis on 1.0% agarose gel,...

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Abstract

The invention belongs to the technical field of bacterial gene engineering of animals, and in particular relates to the construction of a recombinant salmonella choleraesuis strain which does not contain resistance markers and expresses major antigenic loci of porcine transmissible gastroenteritis virus, vaccine preparation and application. In the recombinant salmonella choleraesuis strain C500 (pYA-2SLN) which does not contain the resistance markers and expresses the major antigenic loci of the porcine transmissible gastroenteritis virus, the preservation No. is CCTCC NO: M 209189, and the strain misses asd genes which are necessary for the growth of salmonella choleraesuis and contains plasmid capable of expressing the asd genes, genes of an antigenic locus A, an antigenic locus D and an antigenic locus N321 of the porcine transmissible gastroenteritis virus in the strain. The invention also discloses a method for preparing the salmonella choleraesuis and porcine transmissible gastroenteritis vaccine by utilizing the recombinant strain and application thereof. The prepared recombinant vaccine can stimulate pigs to generate protective immune response for resisting the salmonella choleraesuis and the porcine transmissible gastroenteritis virus, and prevent the infection of the salmonella choleraesuis and the porcine transmissible gastroenteritis virus effectively.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of animal bacteria, and in particular relates to a method for expressing the A and D antigenic sites in the S protein of porcine transmissible gastroenteritis virus and the N in the N protein without a resistance marker. 321 Construction and application of recombinant Salmonella choleraesuis live vaccine strain with antigenic site gene. Background technique [0002] Salmonella choleraesuis (Salmonella choleraesuis) is the main pathogen that causes paratyphoid fever in piglets aged 2-4 months. After infecting pigs, it causes typical symptoms such as acute sepsis, chronic necrotic enteritis, and intractable diarrhea. Infection or secondary infection of other diseases or untimely treatment will lead to a higher mortality rate and cause heavy losses. Animals infected with Salmonella or their products contaminated during their lifetime can cause food poisoning in humans, so this pathogen i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/50A61K39/295A61K39/112A61K39/225A61P31/04A61P31/14C12R1/93C12R1/42
Inventor 何启盖柴伟东宋飞
Owner HUAZHONG AGRI UNIV
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