[Construction and expression of the recombinant plasmid pGEX-Sj14-3-3 of Schistosoma japonicum in BL21(DE3)].
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.


CONCLUSIONS
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.
Institute of Infectious and Parasitic Diseases, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Ning
N
Zhang
Wen-guia
WG
Li
Min
M
Wang
Shi-fei
SF
Cai
D048948:14-3-3 Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012549:Schistosoma japonicum / Q000235:genetics*; D012554:Schistosomiasis japonica / Q000276:immunology
D048948:14-3-3 Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins