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Streptococcus pneumoniae autolysin small fragment gene expression protein, preparation method and application thereof

A Streptococcus pneumoniae, gene expression technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems such as complex operation, increased steric hindrance, and high cost

Inactive Publication Date: 2014-11-19
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of LytA whole protein is not only complicated and costly, but also only some functional groups in LytA whole protein play a role, and the rest may cause side reactions in the body or increase steric hindrance

Method used

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  • Streptococcus pneumoniae autolysin small fragment gene expression protein, preparation method and application thereof
  • Streptococcus pneumoniae autolysin small fragment gene expression protein, preparation method and application thereof
  • Streptococcus pneumoniae autolysin small fragment gene expression protein, preparation method and application thereof

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Effect test

Embodiment Construction

[0026] a. Extract Streptococcus pneumoniae (ATCC49619) genomic DNA;

[0027] b. Take Streptococcus pneumoniae M66 strain in GennBank lyA The full sequence of the gene (FN549899.1), designed upstream and downstream primers: P1 is 5′-GGG GGATCC ATGGAAATTAATGTGAGT-3′, P2 is 5′-GGG AAGCTT TTTTACTGTAATCAAGCCATC-3′, italics are introduced Bam HI and Hind III restriction site;

[0028] With the obtained DNA as a template, the above-mentioned Bam HI and Hind III The PCR reaction primers of the two restriction sites were used for PCR reaction to amplify the target fragment. The PCR reaction conditions were 95°C for 4min; 95°C for 30s, 56°C for 30s, 72°C for 1min, and 30 cycles; 72°C for 7min; % agarose gel electrophoresis identification

[0029] (Such as figure 1 shown), cut the gel to recover the 950bp PCR reaction product;

[0030] figure 1 Middle M: ​​2000 Marker; 1: negative control; 2, 3: target gene; a specific band can be seen at 950bp, consistent with the e...

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Abstract

The invention discloses a streptococcus pneumoniae autolysin small fragment gene expression protein as well as a preparation method and application thereof. The small fragment gene expression protein has the amino acid sequence shown in SEQIDNO:5. The preparation method comprises the steps of extracting the genome DNA of streptococcus pneumoniae (ATCC49619); by taking the obtained DNA as a template, carrying out PCR with a PCR primer having two restriction enzyme cutting sites BamHI and HindIII; splicing the PCR product recycled in glue cutting with a cloning vector to build a clone plasmid; and cutting glue to recycle 500bp small fragments through the clone plasmid built through double digestion respectively by BamHI and HindIII, connecting by a ligase and an expression vector, then converting into a competent cell, picking one and cloning, building a recombination expression plasmid, carrying out IPTG inducible expression, and recycling and purifying the protein. The streptococcus pneumoniae autolysin small fragment gene expression protein is used in preparation of antibacterial medicines.

Description

technical field [0001] The present invention relates to a Streptococcus pneumoniae autolysin small fragment gene expression protein, preparation method and application, especially a Streptococcus pneumoniae ATCC49619 strain autolysin (N-acetylmuramoyl) with simple preparation, low cost and few side effects -L-alanine amidase, LytA) small fragment gene prokaryotic expression protein, preparation method and application in the preparation of antibacterial drugs. Background technique [0002] Streptococcus pneumoniae autolysin (N-acetylmuramoyl-L-alanine amidase, LytA) is one of the cell wall degrading enzymes of Streptococcus pneumoniae, and the whole protein is about 56 kD. At present, the whole LytA protein is mainly used in the vaccine target of Streptococcus pneumoniae and the antigen of serological diagnosis. To obtain the whole LytA protein, it usually needs to go through the following two steps: first, obtain the recombinant protein through prokaryotic expression and nic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/315C12N15/66A61K38/16A61P31/04
Inventor 罗红殷素岚邹琴孙文平杨光谢兴凤周杰吕明睿刘科芳
Owner DALIAN MEDICAL UNIVERSITY
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