【摘要】 目的　将用EST策略及同源性搜索发现的日本血吸虫新基因—Ⅲ 8kDa钙结合蛋白 (Calcium -bindingprotein ,CBP)cDNA克隆到表达质粒pET32a(+)上 ,为下一步对该基因的功能进行研究做准备。方法　将插入于pTriplEx2质粒上的cDNA进行测序 ,测序结果经BLASTn程序搜索 ,发现该插入cDNA序列与曼氏血吸虫钙结合蛋白cDNA高度同源。根据表达质粒pET32a(+)上的克隆位点及该cDNA序列设计PCR引物 ,将PCR产物纯化后连接到pMD 1 8-T载体上 ,将重组T载体经EcoRI/XhoI双酶切后切下的SjCBP基因亚克隆入原核可溶性表达质粒pET32a(+)中。结果　所发现的新基因与曼氏血吸虫 8kDaCBP基因的同源性达 82 % ,PCR产物的片段长度与预期大小一致 ,重组T载体及表达质粒经EcoRI及XhoI双酶切后证明具有与目标片段长度相符的插入片段。结论　所发现的cDNA与曼氏血吸虫 8kDaCBPcDAN高度同源 ,并且已成功地构建出重组表达质粒pET32a(+) -SjCBP。更多还原

【Abstract】 Objective To subclone the novel gene of Schistosoma japonicum -Ⅲ8kDa Calcium binding protein (CBP) cDNA which was discovered in the EST study into the expression plasmid pET32a(+); hence preparing to further research on its function.Method The insert cDNA fragment was sequenced and analyzed with the BLASTn program. This DNA sequence was highly homologous to the Schistosoma mansoni 8kDa Calcium binding protein cDNA. Two PCR primers were designed according to this CBP cDNA sequence and the cloning site in pET32a(+) plasmid. The cDNA was amplified using the designed primers by PCR and subcloned into the pMD18 T vector. The recombinant plasmid was digested with EcoRI and XhoI and the DNA fragment encoding the CBP gene was into the expressing vector pET32a(+).Result The novel gene was highly homologous to the 8kDa SmCBP cDNA with 82% identity. The Schistosoma japonicum CBP cDNA was successfully cloned into the expression vector pET32a(+).Conclusion The novel gene codes for a calcium binding protein of Schistosoma japonicum was identified. The recombinant expression plasmid pET32a(+) SjCBP had been constructed successfully.更多还原