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Construction and expression of lactic acid bacteria for recombination of GP5 gene of porcine reproductive and respiratory syndrome virus

A technology for respiratory syndrome and recombinant lactic acid bacteria, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve problems such as strong virulence, weak immune response, and unsatisfactory immune effects

Inactive Publication Date: 2016-11-09
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is generally believed that the immune effect of the attenuated vaccine is better than that of the inactivated vaccine, which can reproduce in cells and stimulate the body to produce humoral immunity and cellular immunity at the same time, but there may be risks of incomplete inactivation and strong virulence
Inactivated vaccines cannot replicate in cells, the immune response is not strong, and multiple immunizations are required to achieve effective antibody levels, and the immune effect is not ideal

Method used

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  • Construction and expression of lactic acid bacteria for recombination of GP5 gene of porcine reproductive and respiratory syndrome virus
  • Construction and expression of lactic acid bacteria for recombination of GP5 gene of porcine reproductive and respiratory syndrome virus
  • Construction and expression of lactic acid bacteria for recombination of GP5 gene of porcine reproductive and respiratory syndrome virus

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Embodiment 1

[0049] (1) Cloning and sequence analysis of GP5 gene of porcine reproductive and respiratory syndrome virus

[0050] 1. Main test materials and molecular biology reagents

[0051] (1) Sample source

[0052] Suspected PRRSV disease materials were collected from different regions in Yunnan Province, my country, and tissue samples such as lungs, spleen, and lymph nodes were taken, aliquoted into cryopreservation tubes, and stored at -80°C.

[0053] (2) Bacteria and plasmids

[0054] Escherichia coli DH5α and pMD19-T Vector were purchased from Bao Bioengineering (Dalian) Co., Ltd.

[0055] (3) Main materials and reagents

[0056] RNAiso Plus, PrimeScript Reverse Transcriptase, RT-PCR reaction reagents, and DNAMarker were all purchased from Bao Bioengineering (Dalian) Co., Ltd.; DNA gel recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and common plasmid mini kits were purchased From Beijing Quanshijin Biotechnology Co., Ltd.

[0057] (4) Self...

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Abstract

The invention discloses construction and expression of lactic acid bacteria for recombination of a GP5 gene of a porcine reproductive and respiratory syndrome virus and belongs to the technical field of biology. The construction and expression are characterized by comprising the following steps of cloning and sequencing the GP5 gene of the porcine reproductive and respiratory syndrome virus, carrying out dual-enzyme digestion on a recombinant plasmid pMD19(simple)-dGP5 and a carrier pNZ8149 by Nco I and Xba I, connecting purified enzyme-digested products by T4DNA ligases; carrying out electrotransformation on the connected products into competence NZ3900 of lacF gene deletion type lactic acid bacteria, culturing on an LM17 recovery medium, and obtaining recombinant expression plasmid pNZ8149-dGP5 by Ellike plate screening. The construction and expression disclosed by the invention have the beneficial effects that the GP5 gene of the porcine reproductive and respiratory syndrome virus can be recombined in the expression carrier pNZ8149 of a lactococcus lactis; proved by detection, the exogenous gene GP5 can be expressed in the lactic acid bacteria by the carrier pNZ8149 and has reactogenicity, so that an experimental basis is provided for next-step development of safe-effective-cheap oral preparations.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of biotechnology of the construction and expression of a porcine reproductive and respiratory syndrome virus GP5 gene recombinant lactic acid bacteria. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which can cause reproductive dysfunction in sows and Severe respiratory diseases in pigs have brought huge economic losses to my country's pig industry. Porcine reproductive and respiratory syndrome virus particles are spherical, with a diameter of 48-83 nm and an envelope. PRRSV is a single-stranded, positive-strand RNA virus with a genome length of about 15kb, a cap structure at the 5' end, and a poly(A) tail of polyadenylation after the non-coding region at the 3' end. It encodes 9 ORFs, the genes are ORF1a, ORF1b, ORF2a, ORF2b,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/11C12N15/74C12N15/66
CPCC07K14/005C12N15/66C12N15/746C12N2770/10022C12N2800/101
Inventor 张以芳马志亮杨洁周玉照张辰宇郑锦玲刘旭川
Owner YUNNAN AGRICULTURAL UNIVERSITY
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