摘要
目的扩增并表达日本血吸虫表膜蛋白(SjTsp2-A)基因SjTsp2。方法根据与曼氏血吸虫表膜蛋白SmT-sp2基因同源的SjTsp2序列(编号为AY810722)设计引物,体外扩增目的蛋白基因,并将其亚克隆入原核表达载体pET28a,转化至大肠埃希菌(E.coli)BL21株,异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,用纯化的重组蛋白免疫小鼠,ELISA检测其血清抗体效价,蛋白质印迹(Western blotting)分析鉴定其免疫反应性。结果PCR扩增获得SjTsp2基因大环核苷酸序列为228bp,与曼氏血吸虫表膜蛋白(SmTsp2)的氨基酸序列同源性为52%。SjTsp2基因亚克隆入pET28a并可溶性表达,免疫小鼠可产生高滴度(最高1∶32000)特异性抗体。Western blotting分析结果表明,纯化的SjTsp2-A蛋白能被疫区居民血吸虫抗体阳性者血清、急性及慢性血吸虫病患者血清识别,与健康人血清无反应。结论日本血吸虫表膜蛋白基因SjTsp2在体外获得表达,其表达蛋白具有一定的抗原性。
Objective To clone and express a membrane protein （Tetraspanin 2） gene of Schistosoma japonicum （SjTsp2）. Methods A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a（＋）. The recombinant plasmid was transformed into E. coli BL21 （D3） and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. Results Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant （1：32 000 in maximum） was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. Conclusion SjTsp2 has been expressed and shows certain antigenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2008年第1期21-24,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
安徽省自然科学基金(No.050430805)
安徽省教育厅基金(No.2006KJ3586)~~
