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目的克隆和表达肝片吸虫组织蛋白酶L基因(FhCL),分析其免疫原性。方法根据GenBank公布的FhCL基因序列设计引物,以肝片吸虫总RNA为模板,通过RT-PCR扩增FhCL基因编码序列,PCR产物经TA克隆,通过EcoRⅠ、HindⅢ双酶切和测序鉴定获得重组质粒pMD18-T/FhCL,并将其亚克隆入原核表达载体pET30a(+),经PCR,以及BamHⅠ、HindⅢ双酶切和测序鉴定,构建原核表达质粒pET30a(+)-FhCL,转化大肠埃希菌(E.coli)BL21(DE3)pLysS,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并获得纯化的重组蛋白FhCL,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,以及蛋白质印迹(Western blotting)分析该重组蛋白对感染肝片吸虫的山羊血清及免疫的SD大鼠血清的免疫反应性。结果PCR和BamHⅠ、HindⅢ双酶切均可见约1000bp的条带,测序结果显示重组质粒pET30a(+)-FhCL构建成功。SDS-PAGE结果表明,重组蛋白相对分子质量约为Mr42000(含6个组氨酸标签),与目的蛋白相符,以包涵体形式表达。Westernblotting分析结果显示,纯化的重组蛋白FhCL可被感染肝片吸虫的山羊血清和免疫的SD大鼠血清识别,在目的条带Mr42000处见单一特异性条带,而阴性对照血清则无反应带。结论克隆及表达了肝片吸虫组织蛋白酶L编码基因,重组蛋白具有良好的免疫原性。
Objective To clone and express Fasciola hepatica cathepsin L gene (FhCL) and analyze its immunogenicity. Methods The primers of FhCL gene were designed according to the FhCL gene sequence published by GenBank. The FhCL gene coding sequence was amplified by RT-PCR using the total RNA of Fasciola hepatica as a template. The PCR product was cloned by TA and identified by EcoRⅠ, HindⅢ double digestion and sequencing. pMD18-T / FhCL was cloned into prokaryotic expression vector pET30a (+). The prokaryotic expression plasmid pET30a (+) - FhCL was constructed by PCR, restriction endonuclease digestion with BamHⅠand HindⅢ, and transformed into Escherichia coli The recombinant protein FhCL was induced by E. coli BL21 (DE3) pLysS and isopropyl-β-D-thiogalactopyranoside (IPTG), and purified with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and Western blotting were used to analyze the immunoreactivity of the recombinant protein to goat serum infected with Fasciola hepatica and immunized SD rat serum. Results A band of about 1000bp was found by both PCR and BamH Ⅰ and Hind Ⅲ digestion. The sequencing results showed that the recombinant plasmid pET30a (+) - FhCL was successfully constructed. The result of SDS-PAGE showed that the molecular weight of recombinant protein was about Mr42000 (containing 6 histidine tag), which was consistent with the target protein and expressed as inclusion body. Western blotting analysis showed that the purified recombinant FhCL could be recognized by Fetal liver infected goat serum and immunized SD rat sera, with a single specific band at the Mr42000 region of the target band, while no negative band at the negative control serum. Conclusion Cloning and expression of Fasciola hepatica cathepsin L coding gene, the recombinant protein has good immunogenicity.