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Genetic engineering subunit oral bicombined inactivated vaccine

An oral vaccine and subunit technology, which is applied in the field of genetically engineered subunit dual oral vaccines, can solve the problems of toxicity, weak immunogenicity, and strong vaccine immune stress, and achieve strong immunity, morbidity and The effect of reducing the death rate and reducing the economic loss

Inactive Publication Date: 2017-09-15
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a genetically engineered subunit dual oral vaccine, which is a porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus genetically engineered subunit dual oral vaccine. The outstanding advantage of oral immunity is that it can effectively stimulate the intestinal tract Local immune cells produce secretory IgA, which is especially suitable for intestinal mucosal infectious diseases, and avoids the stress and vaccine absorption problems caused by routine injections, and solves the problem of strong immune stress, weak immunogenicity and toxicity of vaccines. question

Method used

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  • Genetic engineering subunit oral bicombined inactivated vaccine
  • Genetic engineering subunit oral bicombined inactivated vaccine
  • Genetic engineering subunit oral bicombined inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: the preparation of oral vaccine

[0021] 1. Synthesis and amplification of the target gene

[0022] According to the RNA sequence of the whole virus, after the antigenic site is optimized, two flexible Linkers ((GGGGS)3) are selected to connect the A and D antigenic site genes of TGEV with the N321 antigenic site gene (the sequence is: D antigen Site gene-Linker-N321 antigenic site gene-Linker-A antigenic site gene), the sequence was named SLN, and sent to the biological company for synthesis.

[0023] Design specific primers to extract RNA from the obtained PEDV mutant strains, amplify the S1 gene of PEDV and the SLN gene of TGEV after reverse transcription and connect to the pMD18-T vector, transform into DH5α-sensitive cells, pick positive single clones, and bacterium liquid PCR identification, extraction plasmid digestion identification and sequencing identification were correct.

[0024] 1. Expression vector construction

[0025] The correctly iden...

Embodiment 2

[0044] (1) Efficacy test of oral vaccine

[0045] 1 material

[0046] 1.1 Drugs

[0047] Recombinant oral vaccine prepared in the laboratory of Qingdao Agricultural University.

[0048] 1.2 Test sites and test animals

[0049] The test site is Haiwei Pig Farm, Chengyang District, Qingdao City, Shandong Province, and the test animals are 20 30-day-old piglets.

[0050] 2 test method

[0051] 2.1 Vaccine immunization and antibody detection

[0052] Twenty 30-day-old piglets were equally divided into control group and immunization group. Each piglet in the immunization group was fed with one dose of combined oral vaccine, and the control group was fed with the same dose of sterile saline. Blood was collected from each pig before immunization and 7d, 14d, 21d, and 28d after immunization, and the antibody content in the blood was measured.

[0053] 2.2 Expression level and law of growth and decline of recombinant bacteria in pigs after vaccine immunization

[0054] In the o...

Embodiment 3

[0064] Embodiment 3: Safety test of oral vaccine

[0065] 1 material

[0066] 1.1 Drugs

[0067] Recombinant oral vaccine prepared in the laboratory of Qingdao Agricultural University.

[0068] 1.2 Test sites and test animals

[0069] The test site is Haiwei Pig Farm, Chengyang District, Qingdao City, Shandong Province, and the test animals are 20 30-day-old piglets.

[0070] 2 test method

[0071] 2.1. Single-dose safety test

[0072] Eighty 30-day-old piglets were divided into two groups, the test group and the control group, with 40 pigs in each group. Each head of the test group was fed with 1ml of recombinant lactic acid bacteria, and the control group was fed with the same dose of sterile saline. After feeding once, the mental state of the piglets was observed for 14 consecutive days. Statistics on production performance such as rate, weight gain, and feed utilization rate.

[0073] 2.2. Single-dose repeated safety test

[0074] Eighty 30-day-old piglets were div...

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Abstract

The invention provides a genetic engineering subunit oral bicombined inactivated vaccine, and in particular relates to a porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus genetic engineering subunit oral bicombined inactivated vaccine, which is prepared by using lactococcus lactis capable of carrying out recombinant expression on S protein of porcine epidemic diarrhea virus and SLN protein of porcine transmissible gastroenteritis virus, wherein an amino acid sequence of the S protein of the porcine epidemic diarrhea virus is SEQ ID No. 1, a nucleotide sequence of a coding gene of the S protein of the porcine epidemic diarrhea virus is SEQ ID No. 2, and the SLN protein is formed by connecting genes of A and D antigenic sites with a gene of an N321 antigenic site of the porcine transmissible gastroenteritis virus. The prepared inactivated vaccine can provide effective homologous attacking protection; after being used for immunization, the inactivated vaccine can generate stronger immunity; after a pig group is inoculated with the vaccine, the morbidity and mortality rates of the pig group are obviously reduced; the genetic engineering subunit oral bicombined inactivated vaccine can be used for effectively preventing the prevalence and spread of porcine epidemic diarrhea and porcine transmissible gastroenteritis and reducing the economic loss, caused by the diseases, of pig husbandry, thus having wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, and in particular relates to a genetic engineering subunit dual oral vaccine. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) of Nidovirales (Nidovirales) family Coronaviridae. It is a highly contagious intestinal infectious disease that occurs frequently in winter and is characterized by high lethality in piglets. Pigs of different ages and different breeds are susceptible, and the incidence rate of suckling piglets, shelf pigs or fattening pigs can reach 100%, especially the most serious harm to suckling piglets, with an average case fatality rate of 50%, which brings serious problems to the pig industry. harm. [0003] Porcine transmissible gastroenteritis (Transmissible Gastroenteritis, TGE) is a kind of porcine transmissible gastroenteritis virus (Transm...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61K39/215A61P31/14C07K14/165C07K14/17C07K19/00C12N15/50C12N15/62C12N1/21C12R1/46
Inventor 单虎商纪娟盖春云张洪亮秦志华刘晓东
Owner QINGDAO AGRI UNIV
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