论文部分内容阅读
目的构建基因工程菌株,获得重组蛋白Sj-FABPc(日本血吸虫脂肪酸结合蛋白)。方法用PCR法从日本血吸虫cDNA文库中扩增Sj-FABPc基因片段,再将该片段重组于pGEMT中并进行DNA测序鉴定,经酶切后将目的片段构建成重组质粒pGEX-6P-1/Sj-FABPc,转化于大肠杆菌BL_(21),IPTG诱导表达。用Glutathione Sepharose~(TM) 4B亲和层析柱对表达产物进行纯化,PreScission~(TM) Protease酶对融合蛋白进行分离,获得纯化的14kDa Sj-FABPc。SDSPAGE和Western blot方法对表达产物进行鉴定。结果获得pGEX-6P-1/Sj-FABPc菌株,分离纯化出14kDa Sj-FABPc,表达量为10.52mg/L。Western blot显示,表达蛋白能被日本血吸虫免疫兔血清识别。结论重组蛋白Sj-FABPc可高效表达并有良好的抗原性。
Objective To construct a genetically engineered strain and obtain the recombinant protein Sj-FABPc (Schistosoma japonicum fatty acid binding protein). Methods Sj-FABPc gene fragment was amplified from the cDNA library of Schistosoma japonicum by PCR. The fragment was then recombined in pGEMT and identified by DNA sequencing. After digestion, the recombinant plasmid pGEX-6P-1 / Sj -FABPc was transformed into E.coli BL_ (21) and induced by IPTG. The expressed product was purified by Glutathione Sepharose 4B affinity chromatography, and the protein was separated by PreScissionTM Protease enzyme to obtain purified 14kDa Sj-FABPc. SDSPAGE and Western blot method to identify the expression product. Results The pGEX-6P-1 / Sj-FABPc strain was obtained and the 14kDa Sj-FABPc was isolated and purified. The expression level was 10.52 mg / L. Western blot showed that the expressed protein could be recognized by Schistosoma japonicum immune rabbit serum. Conclusion The recombinant protein Sj-FABPc is highly expressed and has good antigenicity.