Construction and expression of the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum in Escherichia coli BL21(DE3)
10.3760/cma.j.issn.1000-4955.2011.02.009
- VernacularTitle:日本血吸虫重组质粒pET28α-Sj26GST-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达
- Author:
Wen-gui, LI
;
Bang-zhong, XIAO
;
Xing-jian, LUO
;
Ya-tang, CHEN
;
Cheng-guo, WU
- Publication Type:Journal Article
- Keywords:
Schistosoma japonicum;
Recombinant plasmid pET28α-Sj26GST-Sj32;
Escherichia coli;
Vaccines,synthetic
- From:Chinese Journal of Endemiology
2011;30(2):152-157
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and express the recombinant plasmid pET28α-Sj26GST-Sj32 of Schistosoma japonicum(Sj) in Escherichia coli BL21 (DE3). Methods Total RNA was extracted from Sj adult worms by ultrasound-breaking, Sj26GST and Sj32 antigen gene was respectively amplified by RT-PCR from the total RNA; Sj26GST-Sj32 fusion gene obtained with gene splicing by overlap extension(SOEing) was cloned into prokaryotic expression plasmid pET28α and transformed into Escherichia coli BL2 (DE3) to construct pET28α-Sj26GST-Sj32;BL21 (pET28α-Sj26GST-Sj32) was induced with isopropyl-β-D-thiogalactopyranosid (IPTG), and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blotting. Results The 1991 bp Sj26GST-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into pET28α by restriction analysis and PCR identification, the recombinant plasmid pET28α-Sj26GST-Sj32 was successfully constructed; the relative molecular mass of the expressed recombinant protein was approximately 69 × 103 by SDS-PAGE, and the amount of the expressed protein was 25% of the total bacterial proteins; the fusion protein could be recognized by sera from rabbits infected with Sj by Western blotting.Conclusions The recombinant plasmid pET28α-Sj26GST-Sj32 is successfully constructed and highly expressed in Escherichia coli in fused form with His-tag, and the expressed fusion protein shows specific antigenicity.
