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| Expression and Application Research of the C-terminal Gene Fragment of P1 Protein of Mycoplasma pneumoniae in E.coli |
| HUANG Jin-song1, HUANG Jie-qin2, TANG Qi-hui2, CHEN Yu-bao2 |
1. Beijing Academy of Sciences and Technology, Science and Technology Department,Beijing 100094,China;
2. Beijing Biokit Incorporated, Beijing 100094,China |
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Abstract Objective:To clone, express and characterize the C-terminal gene fragment of P1 protein. Methods:On the basis of successful clone and sequence analysis of the C-terminal gene fragment of P1,the gene fragment were ligated into pET32a(+).An expression vector pET32a(+)/ P1(3 520~4 563bp)were constructed and expressed in E.coli rosetta induced by IPTG. recombinant protein was purified through GSTrap 4B affinity chromatography column. ELISA and Western blotting were used to determine the antigenic of the recombinant protein. Immune mouse with recombination protein, monoclonal antibody was prepared. Results:The recombinant gene can be overexpressed in E.coli. SDS analysis revealed that the expressed protein was identical to C-terminal gene fragment of P1 protein in molecular weight with a purity of 95%. ELISA and Western blotting analysis showed that the recombinant protein can displayed high antigenic activity toward Mycoplasma pneumoniae convalescent antiserum. Two monoclonal antibody cell line were obtained. Conclusion:The C-terminal gene fragment of P1 protein of MP has been successful cloned and expressed, monoclonal antibody was prepared which could be useful for developing diagnose reagents or vaccine of MP.
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Published: 19 November 2010
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