Cloning and expression of Schistosoma japonicum VCP gene and its mRNA expression levels in different stages
- VernacularTitle:日本血吸虫VCP基因的克隆表达及其在不同生活史期的mRNA表达水平
- Author:
Fei WANG
;
Xiaoting WANG
;
Yang DAI
;
Ying XU
;
Yuntian XING
;
Guoli QU
;
Jianrong DAI
- Publication Type:Journal Article
- Keywords:
Schistosoma japonicum;
Valosin-containing protein gene(VCP gene);
Prokaryotical expression;
Fluorescence-based quantitative real-time polymerase chain reaction
- From:
Chinese Journal of Schistosomiasis Control
2014;(2):160-164
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prokaryotically express the valosin-containing protein(VCP)of Schistosoma japonicum,and ana-lyze its VCP mRNA expressions in the cercaria,schistosomulum,adult worm(female and male worms)and egg. Methods RNA of S. japonicum eggs were extracted,and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction(PCR),and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells,and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body,and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The RNA(s)of cercaria,schistosomulum,female adult worm,male adult worm,and egg of S. japonicum were extracted,digested with DNase,purified,and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR. Results The VCP gene of S. japonicum was yielded by PCR amplification,and the recombinant pro-tein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR,while low expressions were found in the schistosomulum,egg,female and male adult worms. Conclusion The recombinant protein encoded by the VCP gene of S. ja-ponicum is successfully obtained,and the VCP mRNA expression is determined in various developmental stages of S. japonicum.
