摘要
目的从原核表达日本血吸虫含缬酪肽蛋白(VCP)基因,并分析其在不同生活史期的mRNA表达水平。方法提取日本血吸虫虫卵RNA,逆转录为cDNA,PCR扩增日本血吸虫VCP基因,亚克隆至原核表达载体pET15b;重组质粒转化入E.coli BL21,异丙基硫代半乳糖苷(IPTG)诱导目的基因表达,并采用包涵体纯化方法获取重组蛋白,产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分析鉴定。提取日本血吸虫尾蚴、童虫、雌虫、雄虫、虫卵RNA,DNase消化,纯化后逆转录为cDNA,采用荧光实时定量PCR分析VCP基因在上述生活史期的表达水平。结果 PCR扩增获得日本血吸虫VCP基因,经重组质粒表达和包涵体纯化成功获得重组蛋白。荧光实时定量PCR检测发现,日本血吸虫VCP基因在尾蚴中的mRNA表达水平最高,在童虫、虫卵、雄虫、雌虫中表达水平较低。结论获得日本血吸虫VCP基因编码的重组蛋白;日本血吸虫VCP基因mRNA在尾蚴阶段表达水平最高,在童虫、虫卵、雄虫、雌虫中表达水平较低。。
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.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
2014年第2期160-164,共5页
Chinese Journal of Schistosomiasis Control
基金
国家自然科学基金(81000749)
江苏省自然科学基金面上项目(BK2010152)
