恶性疟原虫海南株线粒体复合体Ⅱ辅基铁-硫蛋白基因克隆及序列分析

Amplification,Cloning and Sequence Analysis of the Gene Encoding Iron-sulfur Protein(Ip) of Succinate-ubiquinone Oxdoreductase of Plasmodium falciparum Isolate FCC1/HN

目的 构建恶性疟原虫海南株线粒体复合体Ⅱ琥珀酸 泛醌还原酶辅基铁 硫蛋白 (iron sulfurprotein ,Ip)基因原核表达质粒pET2 8α Ip ,测定Ip基因序列 ,为研究铁 硫蛋白基因功能奠定基础。方法 采用PCR技术从恶性疟原虫海南株基因组DNA中扩增出Ip基因 ,扩增产物经纯化后 ,用BamHI +XhoI双酶切 ,定向克隆入pET2 8α质粒 ,转化大肠杆菌DH5α ,再用BamHI+XhoI酶切及PCR扩增对重组子进行鉴定。用Sanger双脱氧链终止法进行DNA序列测定 ,并进行同源性比较。结果 筛选出编码海南株Ip基因的原核表达质粒pET2 8α Ip ,海南株Ip基因序列与K1、3D7株高度同源。结论 pET2 8α Ip重组质粒的构建 ,为进一步研究铁

Objective To construct the prokaryotic expression plasmid pET28α Ip and sequence Ip for the expression Ip protein in vitro.Methods Using polymerase chain reaction (PCR) technique,the gene encoding Ip was specifically amplified from the genomic DNA of Plasmodium falciparum isolate FCC1/HN.The PCR products were purified and digested with BamHI and XhoI.The generated DNA fragment was cloned into plasmid pET28α,and transformed into Escherichia coli( E.coli )strain DH5α.The recombinant plasmids were screened and identified by BamHI and XhoI digestion and PCR amplification.Using the chain termination method,Ip was sequenced.Results The results demonstrated that the recombinant plasmid pET28α contained the exogenous gene encoding Ip from isolate FCC1/HN of Plasmodium falciparum. Comparison Ip sequences between isolate FCC1/HN and K1 and 3D7 shows that there is no difference. Conclusion These finding are important for the expression of Ip in vitro and the study of Ip′s function.