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Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen

A technology for recombinant polypeptides and vaccines, applied in the field of DNA recombination technology and methods in the Ming Dynasty, can solve problems such as autoimmune reactions

Active Publication Date: 2012-10-10
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, traditional bacterial vaccines or subunit vaccines may cause an autoimmune response in the body
So far, there is still no safe and effective group A streptococcal vaccine on the market in the world

Method used

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  • Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen
  • Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen
  • Recombination polypeptide serving as group A streptococcic tetravalent vaccine protective antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Synthesis of recombinant polypeptide gene sequence and construction of Escherichia coli cloning vector

[0042] Download the N-terminal type-specific sequences of four types of M proteins of group A streptococcus emm1, emm3, emm6, and emm18 from the database of the US Centers for Disease Control and Prevention, and obtain the amino acid sequences corresponding to these four types in the recombinant polypeptide; from the report The consensus sequences of the C-terminals of these four types of M proteins were obtained from the literature. The five segments of amino acid sequences were spliced ​​in tandem according to the order of 1-3-6-18-consensus consensus sequence from the N-terminus to the C-terminus (SEQ ID NO: 1).

[0043] Enter the amino acid sequence into the DNAWORK 2.0 online software, and obtain a nucleotide sequence (SEQ ID NO: 2) encoding the recombinant polypeptide with Escherichia coli biased codons after running, and synthesize the nucleotide by using the ...

Embodiment 2

[0055] Construction of recombinant polypeptide expression vector

[0056] BamH I and HindIII double-digested recombinant cloned plasmid PUC18-Strep4 and E. coli prokaryotic expression plasmid PQE30, respectively. Agarose gel electrophoresis to separate the digested fragments, recover and purify the DNA fragments encoding the recombinant polypeptide vaccine and the digested fragments of the PQE30 plasmid, connect and construct the recombinant expression plasmid, named as PQE30-Strep4( Figure 4 ). Escherichia coli M15 was transformed, and the ampicillin and kanamycin double resistance screened positive clones, and the inserted nucleotide sequence was completely consistent with the sequence in SEQ ID NO:3 through sequencing analysis. The obtained engineered strain expressing the recombinant polypeptide was named M15 / PQE30-Strep4.

Embodiment 3

[0058] Expression of Recombinant Peptides

[0059] The engineered bacteria M15 / PQE30-Strep4 obtained in Example 2 was inoculated in 20 ml of LB medium containing 100 μg / ml ampicillin and 25 μg / ml kanamycin, and cultured overnight at 37° C. with vigorous shaking. On the next day, inoculate 20ml of the overnight cultured bacterial solution into 1L LB medium containing 100μg / ml ampicillin and 25μg / ml kanamycin, and culture with vigorous shaking at 37°C until the bacterial solution OD 600 After reaching 0.6, IPTG was added to a final concentration of 1 mM. After continuing to cultivate for 4 hours, collect the bacteria by centrifugation at 4000×g for 20 minutes. Bacterial pellets were analyzed by SDS-PAGE electrophoresis at a band with a high protein expression at a molecular weight of about 24KD ( Figure 5 ). This is consistent with the predicted molecular weight of the expression product. The predicted expression product includes 194 amino acids of the recombinant polypepti...

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Abstract

The invention provides recombination polypeptide, which is prepared by gene recombination technology. The recombination polypeptide is prepared by splicing type-specific amino acid sequences of M1, M3, M6 and M18 serological-type M proteins of rheumatic fever immunogenic group A streptococcus prevailing in the Guangdong province of China, and a common section of conservative amino acid sequence of the C ends of the four serological-type M proteins, wherein the N ends of the four serological-type M proteins comprise protective antigen epitopes and do not comprise human cross reaction epitopes.The recombination polypeptide is experimentally proved to be capable of inducing the generation of bactericidins for the type specificity of the M1, M3 and M6 proteins of the A-group streptococcus soas to be used for preparing recombination polypeptide vaccines for the A-group streptococcus.

Description

technical field [0001] The present invention relates to the field of DNA recombination technology and vaccine medicine, more specifically, the present invention relates to a recombinant polypeptide, which can be used as four serotypes mainly targeting the four serotypes of group A streptococcus prevalent in Guangdong Province, my country, which can cause rheumatic fever. The protective antigen of the valent vaccine, the DNA sequence encoding the recombinant polypeptide, the vector containing the DNA sequence, the host cell containing the vector, the method for preparing the recombinant polypeptide by DNA recombinant technology, and the recombinant polypeptide in the development of group A streptococcal vaccine in the application. Background technique [0002] Group A Streptococcus is one of the most common pathogenic Gram-positive bacteria in clinical practice. Group A streptococci can cause a variety of infectious diseases through air droplets, skin contact, etc. Among all ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/315C12N15/31A61K39/09A61P31/04C12N15/10C12P19/34C12N15/63C12N1/21C12P21/02C12R1/125C12R1/19
Inventor 喻刚杨京生全家妩
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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