华支睾吸虫组织蛋白酶D样天冬氨酸蛋白酶基因的克隆表达和重组蛋白的免疫学分析

目的对新发现的华支睾吸虫(Clonorchis sinensis)的组织蛋白酶D样天冬氨酸蛋白酶基因进行克隆、表达和免疫学初步研究。方法将华支睾吸虫组织蛋白酶D样天冬氨酸蛋白酶基因(去除了信号肽编码序列)克隆到原核表达质粒pET-28a(+)中,在大肠杆菌BL-21/DE3中用IPTG诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白免疫SD大鼠制备抗血清。用蛋白印迹(Western blotting)进行免疫学分析。结果PCR、双酶切及DNA测序结果均表明pET-28a(+)-组织蛋白酶D样天冬氨酸蛋白酶重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠杆菌BL-21/DE3中获得高效表达,经亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能识别感染了华支睾吸虫的SD大鼠血清,表明具有免疫反应性。结论华支睾吸虫组织蛋白酶D样天冬氨酸蛋白酶基因可在原核表达系统中获得具有免疫原性的高效表达,为进一步研究该蛋白的功能奠定了基础。

Cloning and prokaryotic expression of cathepsin D like aspartic protease gene of Clonorchis sinensis and analysis of immunogenicity of the recombinant protein

To clone and express the novel gene named cathepsin D like aspartic protease of Clonorchis sinensis to analyse the immunogenicity of the immunological characteristics recombinant protein. By using the full length cDNA plasmid clone(No.Cs10b01) as template, the coding region of cathepsin D like aspartic protease without signal peptide sequence was amplified by PCR, and cloned into the prokaryotic expression vector pET-28a ( ) and then expressed in E. coli BL21 by IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography. SD rats were immunized with the purified recombinant protein in order to obtain the antiserum and analyze its immunogenicity by Western blotting. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed. The expression products were was purified by Ni-IDA affinity chromatography. The recombinant protein could elicit efficiently specific antibodies in SD rats, indicating its ability of immunogenicity,and could react with the C.sinensis infected rat serum indicating its immunoreactivity. It is concluded that the gene of cathepsin D like aspartic protease of Clonorchis sinensis, can be efficiently expressed in the prokaryotic system with immunological activities.