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Construction method and expression method of coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins

A co-expression carrier and immune protein technology, applied in the field of biogenetic engineering, can solve the problems of inability to form effective protective vaccine antigens, etc., to overcome the insufficient expression of tandem expression, ensure safety and effectiveness, and improve immune protection Effect

Inactive Publication Date: 2014-11-19
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In traditional gene biology, the expression tool can only express one protein, but there are many immune-related proteins in Brucella, and the expression of a single protein cannot form an effective protective vaccine antigen

Method used

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  • Construction method and expression method of coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins
  • Construction method and expression method of coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins
  • Construction method and expression method of coexpression vector of L7/L12, Omp31, Rs alpha and sodC Brucella immune proteins

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Experimental program
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Embodiment 1

[0039] Example 1: Construction and expression of co-expression vector

[0040] 1. PCR amplification of L7 / L12, Omp31, Rsα, sodC coding genes

[0041] Use the bacterial genome extraction kit to extract genomic DNA from the standard strain of Brucella abortus as a PCR template to amplify the required genes. Reaction system: 50μL MasterMix, 2μL upstream and downstream primers, 16μL template, 30μL deionized water; DNA glue The recovery kit recovers four protein coding genes;

[0042] 2. Construction of pETDuet-1-L7 / L12 and pRSFDuet-1-Rsα vectors

[0043] The two vectors, pETDuet-1 and pRSFDuet-1, EcoR I and Not I were double-enzyme digested.

[0044] 40μL digestion system: 4μL of 10×H buffer, 1μL each of EcoR I and Not I, 24μL of carrier, 10μL of deionized water, and 1% agarose gel electrophoresis of digested product and recovered by gel recovery kit.

[0045] EcoRI, NotI double enzyme digestion of L7 / L12, Rsα protein gene 40μL digestion system: 10×H buffer 4μL, EcoR I, Not I each 1μL, targ...

Embodiment 2

[0051] Example 2: Expression detection of the four immune protein co-expression vectors L7 / L12, Omp31, Rsα, SodC First configure the SDS-PAGE solution:

[0052] Tris-glycine buffer: 25mmol / L Tris, 250mmol / L glycine (pH 8.0), 0.1% SDS.

[0053] 2×SDS loading buffer: 100mmol / L Tris.Cl (pH8.0), 200m mol / L mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol.

[0054] Coomassie Brilliant Blue Staining Solution: Dissolve 0.25g of Coomassie Brilliant Blue R250 in 45ml methanol, 45ml water and 10ml glacial acetic acid.

[0055] Decolorizing solution: 30% methanol, 10% glacial acetic acid, distilled water to make up the volume to 100ml.

[0056] Then use the above solution to perform SDS-PAGE detection, including the following steps:

[0057] (1) Wash the glass plate, fix it on the electrophoresis tank, and seal the edge with 2.0% agarose. Prepare 5ml of 15% separation glue according to the formula of molecular cloning, and quickly inject it between two glass plates, with the top of the...

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Abstract

The invention discloses a construction method and an expression method of a coexpression vector of L7 / L12, Omp31, Rs alpha and sodC Brucella immune proteins. The disclosed coexpression vector includes a double expression vector pETDuet-1 and a double expression vector pRSFDuet-1, which are both expressed in the same host bacteria, wherein the two double expression vectors are respectively connected with encoding genes of two Brucella immune proteins. On this basis, the invention also discloses the construction method and the expression method of the coexpression vector. L7 / L12, Omp31, Rs alpha and sodC Brucella immune proteins all with good immunity functions can be expressed simultaneously by the coexpression vector disclosed by the invention and are effectively used for preparing subunit vaccine of the Brucella gene engineering and preventing and controlling various brucellosis.

Description

Technical field [0001] The invention relates to a construction method and an expression method of a co-expression vector of Brucella L7 / L12, Omp31, Rsα, and sodC immune proteins, and belongs to the technical field of biological genetic engineering. Background technique [0002] Brucella is a gram-negative facultative intracellular parasitic bacteria, which widely infects domestic animals, wild animals and humans. Domestic animal Brucella mainly infects sheep, cattle and pigs, among which the pathogenic bacteria to humans Mainly Brucella malta and Brucella abortus; brucellosis infects domestic animals and causes epididymitis in male animals and abortion, placental inflammation and infertility in pregnant domestic animals; for humans, brucellosis can cause acute inflammation And many flu-like symptoms, including wave fever, sweating, back pain, and weakness. In some patients, acute clinical symptoms can last for more than a year, eventually leading to chronic persistent infections...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66A61K39/10A61P31/04
Inventor 周继章曹小安李兆才陈启伟宫晓炜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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