Li Xiao-Juan, Wen Xiao-Bo, Ran Xu-Hua, Li Shu-Dong, Wang Chun-Ren, Piao Fan-Ze
Key Laboratory of Preventive Veterinary Medicine of Heilongjiang Province, Heilongliang August-First Land Reclamation University, Daqing 163319, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2009 Jun;27(3):195-9.
To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein.
Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis.
PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL.
The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.
克隆并表达肝片吸虫组织蛋白酶L样蛋白酶基因（FhCL），并研究重组FhCL蛋白的免疫原性。
根据GenBank中报道的FhCL基因设计特异性引物。以肝片吸虫成虫的总RNA为模板，通过RT-PCR扩增FhCL基因。将PCR产物克隆到pMD18-T载体中，然后亚克隆到pET30a(+)载体中。将重组质粒转化到大肠杆菌BL21(DE3)中，用IPTG诱导蛋白表达。通过SDS-PAGE分析重组FhCL的表达情况。通过Western blotting分析检测其对感染山羊血清和SD大鼠抗FhCL抗血清的免疫反应。
PCR和双酶切显示FhCL基因片段长度约为1000 bp。通过测序鉴定构建的重组质粒pET30a(+)-FhCL。重组蛋白（Mr 42,000）以包涵体形式表达。该蛋白分别被感染山羊血清和用FhCL免疫的大鼠血清识别。
已构建重组质粒pET30a(+)-FhCL，其具有高抗原性。
