重组日本血吸虫亲环素B的克隆、表达及免疫分析

Prokaryotic clone,expression and immunological analysis of recombinant Schistosoma japonicum cyclophilin B

目的克隆、表达日本血吸虫亲环素B(SjCyPB)基因,鉴定并分析重组蛋白的免疫性。方法根据Genbank中日本血吸虫序列设计一对特异性引物,以日本血吸虫cDNA为模板扩增SjCyPB基因,酶切后连接到表达载体pET28,构建pET28a(+)-SjCyPB重组质粒,转化入感受态大肠杆菌BL21/DE3后对质粒进行双酶切和测序鉴定。IPTG诱导表达后,经亲和层析纯化重组蛋白。采用Western Blotting分析鉴定重组蛋白的抗原性。将纯化的重组蛋白免疫大鼠,获得免疫血清,ELISA检测抗SjCyPB特异性抗体滴度。结果构建的pET28a(+)-SjCyPB重组质粒经双酶切和测序鉴定证实SjCyPB基因成功连接到pET28a(+)质粒中。经原核表达后获得纯化的重组蛋白,Western Blotting结果显示,该重组蛋白可与感染日本血吸虫的兔血清结合形成明显的条带。采用ELISA检测重组蛋白免疫后大鼠免疫血清的特异性IgG抗体滴度为1∶51 200。结论成功克隆了日本血吸虫CyPB基因,并获得大量纯化的重组蛋白,重组SjCyPB具有免疫原性和抗原性。

Objective To clone and express Schistosoma japonicum cyclophilin B（SjCyPB）gene in E.coli,and to identify and analyze the immunity of recombinant proteins.Methods A pair of specific primers was designed according to GenBank of Schistosoma japonicum sequence.SjCyPB gene was amplify by PCR and then connected to pET28 vector.The recombinant plasmid pET28a（＋）-SjCyPB was constructed and transformed into E.coli BL21 cell line,the recombinant plasmid was identified by double enzyme digestion and sequence analysis.After induced by isopropyl-B-D-thiogalaetoside（IPTG）,the expressed recombinant protein was purified by affinity-chromatography,and then verified by Western blotting.Rats were immunized recombinant SjCyPB,and the SjCyPB-specific IgG was detected by ELISA.Results SjCyPB gene was successfully inserted into pET28a（＋） vecter which identified by double enzyme digestion and sequence analysis.Recombinant SjCyPB protein was highly expressed in E.coli.The Western Blotting analysis confirmed that the recombinant protein could specifically combine to S.japonicum-infected rabbit serum.Using recombinant protein to immunize rats,the SjCyPB-specific IgG antibody titer was 1∶51 200 detected by ELISA.Conclusion The recombinant SjCyPB is successfully constructed,and recombinant SjCyPB has immunogenicity and antigenicity.