Preparation method of porcine epidemic diarrhea recombinant adenovirus vaccine

A technology of porcine epidemic diarrhea and recombinant adenovirus, which is applied in the field of biological vaccines, can solve the problems of low expression level, poor palatability of tobacco, and inability to modify the prokaryotic expression system, so as to prevent the occurrence of PED and have strong ability Effect

Inactive Publication Date: 2012-06-27
GENIFARM LAB INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some defects in these strategies: for example, the S protein is a glycosylated protein, the prokaryotic expression system cannot be modified accordingly, and the expression level is not high; the palatability of tobacco is not good, and oral immunization cannot be well adopted.

Method used

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  • Preparation method of porcine epidemic diarrhea recombinant adenovirus vaccine
  • Preparation method of porcine epidemic diarrhea recombinant adenovirus vaccine
  • Preparation method of porcine epidemic diarrhea recombinant adenovirus vaccine

Examples

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Embodiment 1

[0024] Example 1 Construction of recombinant adenovirus plasmid pAdeno-S1

[0025] 1.1 Linearization and purification of the recombinant shuttle plasmid pShuttle-CMV-S1 Add in a 0.5mL centrifuge tube: 20.0ul of the recombinant shuttle plasmid, 4.0ul of 10×NE Buffer4, 0.4ul of 100×BSA, 1.0ul of PmeⅠ, add sterilized double distilled water To a total volume of 40.0ul; 37°C for 4.0h, use the DNA gel recovery kit to recover the digested product;

[0026] 1.2 Preparation of BJ5183 Competent Cells Carrying Adenovirus Backbone Plasmid pAdEasy-1 For the method, see molecular cloning;

[0027] 1.3 Transformation of BJ5183 Competent Cells with Linearized Recombinant Shuttle Plasmid Take out BJ5183-AD-1 Competent Cells from the -80°C refrigerator, and after the ice bath melts, add 10ul linearized recombinant shuttle plasmid pShuttle-CMV-S1, mix gently, and place on ice Bath for 30min, then heat stress at 42°C for 90sec, and then ice bath for 5min; add 800μL fresh LB liquid medium preheat...

Embodiment 2

[0030] Example 2 Obtaining of recombinant adenovirus rAd-S1

[0031] 1.1 Linearization and purification of recombinant adenovirus plasmid pAdeno-S1 Add in a 0.5mL centrifuge tube: 30.0ul of recombinant adenovirus plasmid, 6.0ul of 10×NE Buffer1, 0.6ul of 100×BSA, 1.0ul of PacⅠ, add sterilized double distilled water To a total volume of 60.0ul; 37 ℃ water bath overnight, use the DNA gel recovery kit to recover the digested product;

[0032] 1.2 Recovery and subculture of AD-293 cells

[0033] 1.3 Transfect AD-293 cells with recombinant pAdeno-S1 linearized plasmid. Inoculate AD-293 cells on a 12-well cell culture plate at a density of 2×105 one day before transfection, and culture them in a 5% CO2 incubator at 37°C; Transfect when the cells grow to 80-90% full and the cells are in good condition. Discard the old cell culture medium before transfection and replace it with serum-free DMEM cell culture medium preheated at 37°C; in sterile 1.5mL Prepare the following solutions ...

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Abstract

The invention discloses a preparation method of a porcine epidemic diarrhea recombinant adenovirus vaccine. The preparation method provided by the invention comprises the following steps of inserting a DNA sequence of a zone S1 of a porcine epidemic diarrhea virus (PEDV) into an adenovirus shuttle plasmid pShuttle-CMV to obtain pShuttle-CMV-S1, carrying out linearization of the pShuttle-CMV-S1, transforming the linear pShuttle-CMV-S1 into a BJ5183 competent cell containing pAdEasy-1, carrying out homologous recombination, carrying out enzyme digestion, carrying out AD-293 cell transfection, carrying out packaging to obtain a recombinant adenovirus rAd-S1, and carrying out purification, amplification and sub-packaging. After oral immunization, the porcine epidemic diarrhea recombinant adenovirus vaccine can induce generation of mucosal immunity thereby preventing porcine epidemic diarrhea (PED) well.

Description

technical field [0001] The invention relates to the technical field of biological vaccines, in particular to a preparation method of porcine epidemic diarrhea recombinant adenovirus vaccine. Background technique [0002] Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute contagious enteric disease characterized by diarrhea, dehydration and vomiting. Pigs of various ages are all susceptible, especially nursing piglets, the incidence rate is as high as 100%, and the mortality rate is 30% to 90%, which causes great losses to the breeding industry, especially in China. The clinical symptoms and pathological changes are very similar to porcine transmissible gastroenteritis, and differential diagnosis of laboratory etiology is required. [0003] The disease was first reported in the UK in 1971, and it was also reported in my country in the early 1980s. In recent years, the prevalence of PEDV in Europe has become less and less; on the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/225A61P31/14C12N7/01C12N15/50C12N15/63
Inventor 刘德辉何俊
Owner GENIFARM LAB INC
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