摘要
目的 将超抗原金黄色葡萄球菌肠毒素A (staphylococcusenterotoxinA ,SEA)基因与人胎盘碱性磷酸酶(hPLAP 1 )C末端信号肽序列 ,即糖基化磷脂酰肌醇 (glycosyl phosphatidylinositol,GPI)附着信号序列 ,拼接成融合基因 ,并构建该融合基因的真核表达载体。方法 利用基因SOEing法 ,分别扩增hPLAP 1C末端信号肽序列和SEA基因 ,然后将二段基因拼接起来 ,拼接后与pGEM T克隆载体连接 ,再亚克隆到真核表达载体pcDNA3 .1 (+)中 ,测序鉴定。结果 分别成功扩增出hPLAP 1C末端信号肽序列 1 31bp和SEA基因 789bp ,并将二段基因序列成功拼接 (90 1bp) ,且成功构建了真核表达载体pcDNA3 .1 (+) /SEA GPI,经测序是正确的。结论 真核表达载体pcDNA3 .1 (+) /SEA GPI的构建 ,为研究SEA GPI抗肿瘤作用奠定了基础。
Objective To construct mammalian cell expression vector of a chimeric SEA hPLAP 1 cDNA. Methods The sea gene and a DNA fragment encoding the signal for GPI anchor attachment of hPLAP 1 were amplified by PCR. The two amplified gene sequence were annealed to form a chimeric GPI anchored SEA molecule by gene SOEing. The resulting chimera was cloned in pGEM T vector and then sub cloned in pcDNA3.1(+) and verified by sequencing analysis. Results The recombinant mammalian cell expression vector of a chimeric SEA hPLAP 1 cDNA〔pcDNA3.1(+)/SEA GPI〕 was successfully constructed ,the sequence was identical to the designed sequence. Conclusion The recombinant mammalian cell expression vector (pcDNA3.1(+)/SEA GPI) of a chimeric SEA hPLAP 1 cDNA could be successfully constructed ,and the plasmid pcDNA3.1(+)/SEA GPI may foundation for further study of the tumor suppression of SEA GPI.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2002年第5期343-346,共4页
Immunological Journal
基金
浙江省自然科学基金资助项目 (30 1 580和 3991 31 )
