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Construction of Prokaryotic Shuttle Expression Plasmid pBCG-Sj14-Sj26 and Identify Their Expression in Mycobacterium Smegmatis.

Author: ManDanDan
Tutor: DaiWuXing
School: Huazhong University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Schistosoma japonicum Sj14 Sj26 Mycobacterium smegmatis prokaryotic shuttle expression vector
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 8
Quote: 0
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Abstract


In my experiment ,To construct a plasmid containing Sj14 (FABP ) and Sj26(GST) genes(a prokaryotic double expression plasmid) byPCR ,and identify their expression in Mycobacterium smegmatis. Sj14 and Sj26 genes were obtained with pMD-Sj14, and pMD-Sj26 which finished in our laboratory as the template, and cloned into vctor pBCG-SP to construct recombinant plasmid pBCG-SP- Sj14 and pBCG-SP- Sj26. Then two modified gene fragments which encode SD gene fragments and a secreting signal peptide fragments can obtain as the template of pBCG-SP- Sj14 and pBCG-SP- Sj26.And altogether cloned into a prokaryotic shuttle expression vector pBCG2100 , it can also obtain recombinant plasmid pBCG- Sj14 - Sj26. The plasmid was transferred into Mycobacterium smegmatis by electroporation. After it was induced by heat, we observed the expressing efficiency and identified biologic character of Sj14 and Sj26 by SDS-PAGE and western-blot technology.The results showed that the recombinant plasmid pBCG-SP-Sj14 , pBCG-SP-Sj26 and pBCG-Sj14-Sj26 were successfully constructed by PCR results and restriction enzyme analysis. The plasmid was successfully transferred into Mycobacterium smegmatis by electroporation., then we observed the expressing efficiency of Sj14 and Sj26 by SDS-PAGE, and identified biologic character of Sj26 by western-blot technology.The conclusion was that the secreting prokaryotic shuttle expressing plasmid pBCG-Sj14 -Sj26 was constructed successfully , Sj14 protein and Sj26 protein can be expressed in Mycobacterium smegmatis successfully by this vector. It layed foundations for later study of BCG vaccine with Schistosoma japonicum.

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