【摘要】 目的　探讨日本血吸虫疫苗候选分子 -钙离子激活的中性蛋白激酶 (Calpain)在抗日本血吸虫感染的保护性作用及其保护性免疫机制。方法　从日本血吸虫成虫中提取RNA ,用RT -PCR扩增Calpain含多个B ,T细胞表位的片段 ,引物中包含BamHI和EcoRI的酶切位点 ,PCR扩增产物经纯化后TA克隆 ,转化的阳性TA克隆经PCR筛选后 ,液体培养大肠杆菌并回收质粒DNA ,质粒DNA经BamHⅠ和EcoRⅠ双酶切 ,目的基因亚克隆到真核表达质粒pVAC载体 ,构建 pVAC -Cal pain真核表达体系 ,转化的阳性亚克隆经PCR筛选 ,液体培养大肠杆菌并回收pVAC -Calpain质粒DNA ,质粒DNA经BamHⅠ和EcoRⅠ双酶切和DNA序列分析鉴定被亚克隆的Calpain基因。 结果　RT -PCR从日本血吸虫RNA中扩增了 4 5 3bp的Calpain基因 ,经BamHⅠ和EcoRⅠ双酶切和DNA序列分析鉴定Calpain基因被克隆到真核表达质粒 pVAC载体。 结论　日本血吸虫疫苗候选分子Calpain的DNA疫苗体系的建立将有助于解析这个疫苗候选分子抗日本血吸虫感染的保护性免疫作用及保护性免疫机制。更多还原

【Abstract】 Aim To explore protection immunity and protection immune mechanism against infection of S japonicum,a calcium activated neutral proteinase(Calpain)as a vaccine candidate molecule Methods RNA was isolated from S japonicum adult,Calpain fragment which contain B and T epitopes was amplified by RT PCR,and PCR product was TA cloned and selected by PCR,then,subcloned into pVAC eukaryatic express vector,and pVAC Calpain express system was isolated from positive clone of culture,goal gene was identified by the way of enzyme digestion of BamHⅠ and EcoRⅠ and DNA sequence analysis from isolated pVAC Calpain Results A 453bp fragment of Calpain was amplified from S japonicum RNA,and the results of enzyme digestion and DNA sequence analysis showed Calpain gene was subcloned into pVAC eukaryatic express vector Conclusion Preparation for Calpain DNA vaccine of S japonicum will account for the protection immunity and protection immune mechanism of the Calpain vaccine candidate molecule更多还原