Ning Zhang1, Wen-guia Li, Min Wang, Shi-fei Cai. 1. Institute of Infectious and Parasitic Diseases, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Abstract
OBJECTIVE: To construct and research the expression of the recombinant plasmid pGEX-Sj14-3-3 in Escherichia coli BL21 (DE3). METHODS: Sj14-3-3 gene was amplified by RT-PCR from template of the total RNA extracted from adult worms of S. japonicum, and then cloned into the vector pGEX-1gammaT to construct pGEX-Sj14-3-3. The recombinant plasmid pGEX-Sj14-3-3 was transformed into E. coli BL21 (DE3). BL21 (pGEX-Sj14-3-3) was induced with isopropyl-beta-D-thiogalactopyranosid (IPTG), and the expressed products were identified by SDS-PAGE and Western blot. RESULTS: A 399 bp fragment of Sj14-3-3 coding gene was successfully amplified by RT-PCR and cloned into the vector pGEX-1gammaT, and the recombinant plasmid pGEX-Sj14-3-3 was constructded successfully. The molecular mass of the expressed recombinant protein was proximately 40 000 Dolton as detected by SDS-PAGE. The amount of the expressed protein was about 21% of the total bacterial protein. Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum. CONCLUSION: The recombinant plasmid pGEX-Sj14-3-3 was successfully constructed. The Sj14-3-3 protein was highly expressed in E. coli and the expressed recombinant protein possessed specific antigenicity.
OBJECTIVE: To construct and research the expression of the recombinant plasmid pGEX-Sj14-3-3 in Escherichia coli BL21 (DE3). METHODS: Sj14-3-3 gene was amplified by RT-PCR from template of the total RNA extracted from adult worms of S. japonicum, and then cloned into the vector pGEX-1gammaT to construct pGEX-Sj14-3-3. The recombinant plasmid pGEX-Sj14-3-3 was transformed into E. coliBL21 (DE3). BL21 (pGEX-Sj14-3-3) was induced with isopropyl-beta-D-thiogalactopyranosid (IPTG), and the expressed products were identified by SDS-PAGE and Western blot. RESULTS: A 399 bp fragment of Sj14-3-3 coding gene was successfully amplified by RT-PCR and cloned into the vector pGEX-1gammaT, and the recombinant plasmid pGEX-Sj14-3-3 was constructded successfully. The molecular mass of the expressed recombinant protein was proximately 40 000 Dolton as detected by SDS-PAGE. The amount of the expressed protein was about 21% of the total bacterial protein. Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum. CONCLUSION: The recombinant plasmid pGEX-Sj14-3-3 was successfully constructed. The Sj14-3-3 protein was highly expressed in E. coli and the expressed recombinant protein possessed specific antigenicity.