Preparation and application of brucella gene engineering subunit vaccines

A Brucella, subunit technology, applied in bacteria, fungi, antibacterial drugs, etc., can solve problems such as no vaccines

Active Publication Date: 2012-07-04
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no vaccine against the genetically engineered subunit of Brucella. In order to e...

Method used

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  • Preparation and application of brucella gene engineering subunit vaccines
  • Preparation and application of brucella gene engineering subunit vaccines
  • Preparation and application of brucella gene engineering subunit vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 The source of the fusion protein gene

[0049] The coding gene of the entire fusion protein is artificially designed according to the preferred codons of Escherichia coli, and is named as the ORF-BLS fragment (it has a sequence of 1-969 nucleotide sequences in SEQ ID No.1 and submitted to Shanghai Handsome Biotechnology Co., Ltd. Synthesized by the company, the nucleotide sequence at position 970-1035 in SEQ ID No.1 is provided by the nucleotide sequence at position 1277-1340 of the vector pPICZalpha A of Invitrogen Company. EcoRI ( 5' end) and XbaI (3' end) restriction endonuclease sites. Shanghai Handsome Biotechnology Co., Ltd. synthesized this fragment and cloned it into the pMD18T vector, transformed Escherichia coli, extracted and sequenced correctly, and named this fragment as The Escherichia coli of the pMD18T-ORF-PBLS recombinant plasmid was sent to our company.

Embodiment 2

[0050] Example 2 Construction of Fusion Protein Yeast Expression Vector

[0051] The Escherichia coli strain containing the recombinant plasmid named pMD18T-ORF-PBLS was inoculated into the 10mlLB medium containing ampicillin 50mg / L, and the Escherichia coli strain containing the Pichia pastoris secretion expression vector pPICZalphaA (purchased from Invitrogen Company), Inoculate into low-salt LB medium containing 25mg / L Zeocin, culture overnight at 37°C with shaking, and extract plasmids respectively according to the instruction manual of Qiagen plasmid extraction kit the next day. Recombinant plasmids containing genes encoded by fusion fragments were treated with restriction endonucleases corresponding to both ends of the fragments, and pPICZalpha A, a secretion expression vector of Pichia pastoris, was treated with EcoRI and XbaI. Specific treatment conditions: 10l reaction system, 2l plasmids were added to the system , each restriction endonuclease used 5 activity units (...

Embodiment 3

[0055] Example 3 Construction and Screening of Fusion Protein Expression Strains

[0056] Inoculate the above-mentioned positive clones into 50ml low-salt LB medium containing 25mg / L Zeocin. After 8-12 hours, transfer to 500-1000ml low-salt LB medium containing 25mg / L Zeocin, cultivate overnight, and extract a large number of plasmids for later use. .

[0057] Preparation of linearized DNA: In a 100 l reaction system, add 20 g of the DNA extracted above, add Pme I 20∪ (New England biolabs), and make up with deionized water. Digest at 37°C for 3 hours. Take 2 liters of digested products and run them on 1% agarose gel electrophoresis to observe whether the digested enzymes are complete. After confirming that the linearization is complete, add 2l of 200mM EDTA to the remaining digested product to terminate the reaction.

[0058] Purification of linearized DNA: Add 100l of deionized water to the above enzyme digestion system, add an equal volume of phenol / chloroform, shake vigo...

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PUM

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Abstract

The invention relates to a fusion protein for preventing brucella infection and a preparation method and application of the fusion protein. In particular, the invention relates to a fusion protein, which contains brucella L7/L12 protein ORF, a subunit of a brucella BLS protein, a T cell antigen epitope and a purification tag. The invention also relates to the preparation method and the medicinal application of the fusion protein.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to a fusion protein used for immunity, which is used to prevent the infection of Brucella bovis, which consists of a subunit of Brucella L7 / L12 protein ORF, Brucella BLS protein, Composition of T cell antigen epitope and purification tag sequence, preparation method and application thereof. Background technique [0002] Brucellosis (Brucellosis), referred to as brucellosis, is a zoonotic infectious allergic disease caused by Brucella (Brucella). In 1886, British military doctor Bruce isolated and cultured the bacterium from the spleen of a soldier who died of Malta fever on the island of Malta, which was then called Micrococcus melitensis. Most types of Brucella bacteria are pathogenic to humans and animals, and Brucella can enter the body through the skin, respiratory tract and digestive tract to infect. After entering the body, it first invades the local lymph ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P21/02A61K39/10A61P31/04
Inventor 李殿明蒲勤李毅赵明田春辉齐春梅顾富香
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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