恶性疟原虫FCC1/HN株嘌呤核酸磷酸化酶(PNP)基因的克隆和原核表达

Cloning and expression of the gene of Plasmodium falciparum FCCI/HN encoding purine nucleoside phosphorylase

目的 从恶性疟原虫基因组中扩增、克隆恶性疟原虫嘌呤核酸磷酸化酶(PNP)基因,并进行原核表达产物。方法根据恶性疟原虫FCB株嘌呤核酸磷酸化酶基因编码序列,设计一对引物,引入EcoR I和Xho I。采用PCR技术从恶性疟原虫FCC/HN株基因组DNA中特异扩增PNP基因。纯化后扩增产物用EcoR I和Xho I双酶切后,定向克隆入原核质粒pET30a(+)和真核质粒pcDNA3,重组质粒pET30a(+)-PNP转化大肠杆菌BL21(DE3),筛选阳性重组子后,用PCR、EcoRI+Xho I双酶切和DNA序列测定鉴定。用IPTG诱导重组质粒pET30a(+)-PNP表达融合蛋白。结果 从恶性疟原虫FCC1/HN株基因组中特异扩增出PNP基因,将扩增的目的基因亚向插入pET30a(+)和pcDNA3表达质粒的EcoR I和XhoI位点;重组子pET30a(+)-PNP在大肠杆菌BL21(DE3)诱导表达中表达,表达融合蛋白分子量为31.4kDa。结论 从恶性疟原虫基因组中获取PNP基因,并成功构建pET30a(+)-PNP和pcDNA3-PNP重组质粒,获得PNP原核表达产物。

Amplification, cloning and expression of the gene encoding the purine nucleoside phosphorylase (PNP) of Plasmodiun falciparum FCC1/HN, isolated from the southern China isolate. According to the published PNP gene sequence from Plasmodi-um falciparum, a pair of oligonudeotides was designed as primers, the gene of the purine nucleoside phosphorylase of the FCC1/HN isolate of Plasmodium falciparum was amplified by using PCR technique.The PCR product was purified and digested with EcoR 1 and Xho I , then cloned into the plasmid pET30a( + ) and pcDNA3 at the EcoR I and Xho I sites. The recombinant plasmid were i-dentified by restriction analysis, PCR amplification and DNA sequence analysis, then the recombinant plasmid pET30a( + )-PNP was transformed into E.coli BL21(DE3) .The recombinant pET30a( + )-PNP was induced by IPTG concentration. A high level expression of PNP fusion protein was analyzed. The results showed that the gene of PNP was amplified from the genome of Plasmodiun falciparum FCC1/HN, the southern China isolate and the purified PCR product was directly inserted into the plasmids pET30a( + )and pcDNA3 at the EcoR I and Xho I sites.The PNP fusion protein induced by IPTG concentration was analyzed by SDS-PAGE, the fusion protein molecular weight is 31.4kDa. It concludes that PNP gene is obtained from genome of Plasmodium falciparum and the recombinant plasmids of the pET30a( + )-PNP and pcDNA3-PNP are successfully constructed .The PNP fusion protein expresses in E. coli BL21(DE3) and analyzed by SDA-PAGE.