Recombinant expression and use for pertussis vaccine protective antigen

A pertussis and vaccine technology, applied in the field of bioengineering, can solve the problems of limited yield of active ingredients, antigenic variation, complex purification process, etc., and achieve the effect of improving immune protection effect.

Inactive Publication Date: 2007-11-21
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another object of the present invention is to use the pertussis genetic engineering vaccine developed and produced by genetic engineering technology to contain the above-mentioned recombinant PRN, as well as recombinant FIM2 and FIM3, which can solve the problem of using pertussis culture supernatant to purify the production of active ingredients in the antigen preparation method Limited, complex purification process, antigenic variation and other issues

Method used

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  • Recombinant expression and use for pertussis vaccine protective antigen
  • Recombinant expression and use for pertussis vaccine protective antigen
  • Recombinant expression and use for pertussis vaccine protective antigen

Examples

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Comparison scheme
Effect test

Embodiment 1

[0073] Cloning of embodiment 1., PRN, FIM2 and FIM3 genes

[0074] 1.1 Amplification of PRN, FIM2 and FIM3 genes

[0075] Pertussis genomic DNA was extracted from B. pertussis CS strain (preserved in the serum laboratory of China National Institute for the Control of Pharmaceutical and Biological Products) with a whole genome DNA extraction kit (Promega). Primers were designed using PrimerPremier 5.0 software, and according to the pertussis PRN, FIM2 and FIM3 gene sequences reported by GeneBank (GenBank accession numbers are J04560, Y00527, X51543): respectively design 3 pairs of primers as shown in Table 1:

[0076] Gene

Primer sequence

(5'-3')

Location

(nt)

length

(bp)

PRN

FIM2

701

FIM3

Upstream 5'-ATGGCGGGCGTCTGCTCTCCACCTG-3'

Downstream 5-ACCTTGAAGTCATTCTCCAGGCGGC-3′

Upstream 5′-ACCCATGCAAATCCCTTTTCCAACGC-3′

Downstream 5′-GTATGTTGGCGATTTCCAGTTTCTC-3′

Upstre...

Embodiment 3

[0086] Example 3. Construction of recombinant expression plasmids

[0087] Target genes were amplified by PCR, and corresponding primers were designed according to the pertussis PRN, FIM2 and FIM3 gene sequences reported by GeneBank (GenBank accession numbers are J04560, Y00527, and X51543, respectively). The designed primers were used to amplify PRN, FIM2 and FIM3 genes respectively, and construct the corresponding expression plasmids of PRN, FIM2, FIM3 and FIM2-FIM3 fusion genes. Referring to Figures 2-1, 2-2, 2-3, and 2-4, the constructed prokaryotic expression plasmids are PRN-PQE30, FIM3-PQE30, FIM2-PET30a and FIM2-FIM3-PET30a.

Embodiment 4

[0088] Example 4. Expression of recombinant protein in Escherichia coli

[0089] The constructed recombinant plasmids were introduced into Escherichia coli to induce expression, that is, the recombinant plasmids PRN-PQE30 and FIM3-PQE30 were transferred into E. coli M15, FIM2-PET30a and FIM2-FIM3-PET30a were transferred into E. coli BL21(DE3), IPTG induced the expression of the target protein PRN, FIM2, FIM3 and FIM2-FIM3 fusion protein, and then purified the target protein; then analyzed by SDS-PAGE.

[0090] 4.1 Expression levels of recombinant proteins FIM2, FIM2-FIM3, FIM3 and PRN

[0091] SDS-PAGE gel scanning analysis was performed on the expression products, and ImageMaster TotalLab software analyzed and determined the protein expression level. The expression levels of the target proteins PRN, FIM2 and FIM2-FIM3 accounted for more than 40% of the total bacterial protein, and the expression level of FIM3 was 25%. See Figures 3, 4, 5, and 6.

[0092] 4.2 Existing forms...

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Abstract

A method for cloning PRN FIM2 and FIM3 genes from Bordetella pertussis CS strain and prokaryotic expressed recombinant protein by expression carrier are disclosed. The procedure is carried out by cloning CS strain to obtain PRN, FIM2 and FIM3 genes, sub-cloning to expression carriers separately, constructing FIM2-FIM3 fused gene expression plasmid, inducing and expressing in colibacillus. It has excellent immune protection and immunogenicity. They can be used as pertussis vaccine antigen active ingredients.

Description

field of invention [0001] The invention relates to the field of bioengineering, in particular to obtaining the protective antigen protein of bacillus pertussis through genetic engineering and using it for the production of pertussis vaccine. Background technique [0002] Pertussis is a serious respiratory infectious disease caused by Bacillus pertussis. Pertussis vaccine is the most effective and economical way to prevent pertussis. According to the statistics of the World Health Organization (WHO), there are still 35 million pertussis patients in the world every year, of which up to 325,000 children die from pertussis and its complications, and 90% of the cases come from underdeveloped and developing countries. In recent years, even in countries with high vaccine coverage, there is still a trend of rising incidence, local outbreaks, and the number of pertussis cases in adults and adolescents has also increased sharply, which is called "re-emergence of whooping cough". )". ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/195A61K39/10A61P11/00C07K14/235
Inventor 王雅英侯启明徐颖华谭亚军
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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