人乙酰胆碱酯酶(AchE)cDNA克隆及真核表达载体的构建

目的　克隆人乙酰胆碱酯酶 (AchE)cDNA并构建真核表达载体。方法　根据GeneBank中hAchE核苷酸序列 ,设计并合成 1对特异性扩增hAchE的引物 ,从 18周龄引产的胎儿大脑组织中提取RNA为模板 ,利用RT PCR技术扩增出hAchEcDNA片段 ,并与pGEM T载体连接 ,转化大肠杆菌DH5α中 ,经IPTG X gal诱导培养 ,筛选白斑提取重组载体 ,经酶切和PCR鉴定及序列测定与分析 ,并构建hAchE真核表达载体pcDNA3 .1(+) hAchE。结果　已克隆的hAchEcDNA片段全长约 1.9kb ,其测序结果与基因文库中的人乙酰胆碱酯酶cDNA序列相符。结论　在国内首次利用RT PCR成功地克隆了hAchEcDNA全长 ,并构建了hAchE真核表达载体pcDNA3 .1(+) hAchE ,为进行hAchE的结构与相关功能研究和基因工程生产奠定了基础。

Cloning of Human Acetylcholinesterase (AchE)cDNA and Construction of Eukaryotic Expression Vector

Objective To clone human acetylcholinesterase(hAchE) cDNA and construct an eukaryotic expression vector.Methods Design and synthesize a pair of primers according to the sequence of hAchE in GeneBank,and amplify a hAchE cDNA fragment by RT PCR,using the RNA extracted from the brain tissue of induced fetus at 18 weeks of age as a templet.Insert the cDNA fragment into pGEM T vector and transform to E.coli DH5α and express under induction of IPTG/X gal.Select white bacterial colonies,extract recombinant plasmid,identify by restriction endonuclease,PCR and sequencing,and construct eukaryotic expression vector pcDNA3.1(+) hAchE.Results The full length of cloned hAchE cDNA was about 1.9kb,and the sequence of it was identical to that of hAchE cDNA in GeneBank.Conclusion The full length of hAchE cDNA was cloned by RT PCR at first in China,and the eukaryotic expression vector pcDNA3.1(+) hAchE was successfully constructed.It laid a foundation of study on the structure and function,as well as preparation of hAchE by genetic engineering technique.