恶性疟原虫FCC—1／HN株环子孢子蛋白基因分枝杆菌——大肠杆穿梭表达重组质粒的构建及序列测定

本文对恶性疟原虫环子孢子蛋白(circumsporozoiteprotein,CSP)基因片段进行克隆和序列测定。根据恶性疟原虫837株基因编码序列设计合成一对引物，采用PCR技术从恶性疟原虫FCC-1/HN株基因组DNA中特异扩增CSP基因片段的Ⅰ区、中央重复区、重复区后可变区和Ⅱ区；经纯化的扩增产物用BamHⅠ和KpnⅠ双酶切后，定向克隆入大肠杆菌——分枝杆菌穿梭表达质粒，转化感受态大肠杆菌DH5α，重组克隆经抗性筛选和快速凝胶电泳鉴定，再经PCR和酶切鉴定，并对重组子进行序列测定。结果表明从恶性疟原虫FCC-1／HN株基因组DNA中可特异扩增出约1171bp的基因片段，阳性重组质粒经双酶切和PCR鉴定与预期的结果一致，序列测定表明所克隆的基因和编码环子孢子抗原的基因片段相符。

OBSTRUCTION AND SEQUENCE DETERMINATION OF E.COLI-MYCOBACTERIA RECOMBINANT SHUTTLE PLASMID OF CSP GENE FRAGMENT OF PLASMODIUM FALCIPARUM

The circumsporozoite protein(CSP) gene fragment of Plasmodium falciparum was cloned and sequenced.A pair of primer was designed according to the CSP encoding sequence of 837 isolate(Thailand isolate),and the CSP gene fragment was amplified by polymerase chain reaction(PCR) from Plasmodium falciparum FCC-1/HN isolate,it spanned the conserved region I.the central immunodominant repeat region\,the variable region behind the repeats and the conserved II region.After purification,the CSP gene fragment was digested with restriction enzyme BamH I and Kpn I,and ligated with the pBCG5.6 digested with the same enzyme.The recombinant pBCG5.6/CSP was transformed into E.coli DH5α.Positive clones were screened and identified by PCR technique and digestion with restriction enzyme.Nucleotide sequence was determined by dideotide chain-termination method.The results indicated that the CSP gene was successfully amplified and cloned,its nucleotide sequence was about 1 171 bp and was in accordance with the expected one.Sequence determination results showed that the cloned gene fragment was the same with the CSP encoding gene.