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目的 由华支睾吸虫成虫cDNA文库中筛选重要新基因并进行功能基因研究.方法 生物信息学、基因克隆表达技术、免疫识别、S-P免疫组化等方法.结果 由华支睾吸虫成虫cDNA文库成功分离到了CsRPEF(RNA聚合酶Ⅱ延长因子)基因.通过BLASTX程序同源性比对,显示CsRPEF基因与小鼠和人类的RPEF基因同源性为82%.成功构建了CsRPEF原核表达载体,并在大肠埃希菌中进行了大规模诱导表达,所表达的GST融合蛋白经蛋白酶原切割后获得CsRPEF重组蛋白.凝胶扫描分析和Bradford法显示,CsRPEF重组蛋白的纯度和浓度分别为85%和(0.73±0.02) mg/ml.将CsRPEF重组蛋白接种至BALB/c小鼠进行免疫实验.间接ELISA法显示免疫小鼠抗体滴度达1∶150.免疫识别结果显示此抗体可识别20 ku CsRPEF重组蛋白和华支睾吸虫20 ku虫源性蛋白.S-P免疫组化方法显示CsRPEF主要定位于华支睾吸虫成虫的表皮、皮下组织和虫卵三部位.CsRPEF新基因序列GenBank的登录号为EU232119.结论 CsRPEF基因是防治华支睾吸虫病生物药物研发的重要靶点.“,”Objective Clonorchiasis sinensis is caused by infection of Clonorchis sinensis. RNA polymeraseⅡ elongation factor in C. sinensis (CsRPEF) has been considered to play an important role in multiple regulation of cell function via control of transcription elongation of the parasite. However, the gene and protein structures of CsRPEF have not been clarified yet. Methods Bioinformatics, gene cloning, expression, and S-P immunohistochemistry analysis were adopted. Results The present study successfully isolated the CsRPEF gene from a C. sinensis cDNA library. DNA sequencing and BLASTX program homology comparison showed that CsRPEF has 82% homology with RNA polymeraseⅡ elongation factor of mouse and human. The recombinant CsRPEF was expressed in E. coli BL21 by construction and induction of pGEX-4T-RPEF expression vector. The purified recombinant CsRPEF (85% pure) was used to immunize mice and the antiserum obtained were subjected to Western blotting and immunohistochemical analysis. Western blotting analysis showed that the antisera could recognize a 20 ku protein in extract of C. sinensis as well as 20 ku recombinant CsRPEF protein. S-P immunohistochemical analysis suggested that CsRPEF was mainly expressed in epidermis, hypodermics and egg of C. sinensis. Conclusion The present data will be useful to screen potential drug targets and diagnosis candidate antigens for C. sinensis. The nucleotide sequence reported in this paper has been registered to the GenBank database with accession number EU232119.