恶性疟原虫海南FCC1/HN株环子孢子蛋白(CSP)基因的克隆与表达

目的　为疟疾疫苗的研制提供靶抗原。方法　根据恶性疟原虫ＩＭＴＭ２２ 株７Ｇ８ 克隆环子孢子蛋白基因编码区序列， 设计一对引物， 采用ＰＣＲ技术从恶性疟原虫ＦＣＣ１／ＨＮ株基因组ＤＮＡ中特异扩增ＣＳＰ基因的Ⅰ区、中央重复区和Ⅱ区片段， 全长１－０８ｋｂ； 纯化扩增产物用ＨｉｎｄⅢ和ＢａｍＨ Ⅰ双酶切后， 定向克隆入ｐｃＤＮＡ３ 载体， 转化大肠杆菌ＴＧ１ 株， 重组克隆经筛选后， 用ＰＣＲ 扩增和ＨｉｎｄⅢ＋ ＢａｍＨ Ⅰ双酶切进行鉴定： 用磷酸钙贴壁细胞转化法将重组质粒ｐｃＤＮＡ—ＣＳＰ导入ＨｅＬａ细胞， 用Ｇ４１８ 筛选出稳定分泌ＣＳＰ抗原的阳性细胞克隆； 将阳性细胞克隆系扩大培养并用Ｇ４１８ 加压， 以表达重组ＣＳＰ， 分离细胞培养上清和培养细胞， 进行ＳＤＳ ＰＡＧＥ分析。结果　（１） 从ＦＣＣ１／ＨＮ株基因组ＤＮＡ中特异扩增出ＣＳＰ基因Ⅰ区， 中央重复区和Ⅱ区编码序列；（２） 将扩增的目的片段正向插入ｐｃＤＮＡ３ＨｉｎｄⅢ和ＢａｍＨⅠ位点； （３） 在人宫颈癌细胞系ＨｅＬａ 细胞中表达重组ＣＳＰ抗原， 其分子量为３８－３ｋＤａ， 蛋白扫描分析表达量占细胞培养上清液中蛋白总含量的１５－７７％ 。结论　成功构建真核表达系统ｐｃＤＮＡ３

CLONING AND EXPRESSION OF THE GENE ENCODING THE CIRCUMSPOROZOITE PROTEIN OF PLASMODIUM FALCIPARUM, THE SOUTHERN CHINA ISOLATE FCC1/HN

Aim To provide the target antigent for the development of a malaria vaccine Methods According to the published nucleotide sequence of the 7G8 clone of the IMTM22 isolate of Plasmodium falciparum from Brazil,a pair of oligonudeotides was designed as primers The gene coding for the regions Ⅰ to Ⅱ in circumsporozoite protein(CSP)of P falciparum isolate FCC1/HN(1 08kb)has been amplified using PCR technique The PCR product was purified and digested with Hind Ⅲ and BamH Ⅰ then cloned into the plasmid pcDNA3 at the HindⅢ and BamHⅠ sites The recombinant plasmid pcDNA3 CSP was identified by restriction analysis and PCR amplification and transformed into HeLa cell line using calcium phosphate DNA coprecipitation method Transformants were cultured in selective medium containing 200μg of antibiotic G418/ml Clones were subcultured under the high pressure condition of G418(200 to 800 μg/ml) The supernantant of cultures and cells were collected to be analyzed by SDS PAGE Results(1)The gene fragment encoding the regions Ⅰ to Ⅱ in CSP from P falciparum isolate FCC1/HN was successfully amplified;(2)The purified PCR product was directly inserted into the HindⅢ and BamHⅠ site of plasmid pcDNA3;(3)The stable expression of the regions Ⅰ to Ⅱ in CSP was obtained in two clonies of C5 and C11,and the amount accounts was up to 15 6% of the total proteins in the cultured supernantants The molecular weight of the expressed protein is about 38 3kDa on the PAGE,similar to the deduced value(37 4kDa) Conclusion The eukaryotic expression system pcDNA3 CSP/Hela has been established Two clonies C5 and C11 were obtained to be able to secret the recombinant CSP These findings may pave the way to observe the immumogenicity or protective immune responses induced by this expressed protein