恶性疟原虫FCC1/HN株Pf12基因体外扩增、克隆及序列分析

AMPLIFICATION,CLONING AND SEQUENCE ANALYSIS OF THE GENE ENCODING SURFACE MEMBRANE ANTIGEN Pf12 OF PLASMODIUB FALCIPARUM ISOLATE FCC1/HN

目的 构建恶性疟原虫FCC1/HN株Pf12基因原核表达质粒pET2 8α -Pf12 ,测定Pf12基因序列 ,并为FCC1/HN株Pf12的体外表达奠定基础。方法 采用PCR技术从恶性疟原虫FCC1/HN株基因组DNA中扩增出Pf12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 ,并应用PCGENE软件进行同源性比较及预测抗原表位。结果 筛选出编码FCC1/HN株Pf12基因的原核表达质粒 pET2 8α -Pf12 ,FCC1/HN株Pf12基因序列与FMG株高度同源 ,Pf12基因序列中可能存在抗原表位。 结论 pET2 8α-Pf12重组质粒的构建 ,为恶性疟原虫FCC1/HN株Pf12基因的体外表达奠定基础。

Aim Constructing the prokaryotic expression plasmid pET28 a-Pf12 sequencing Pf12 and expressing Pf12 protein.Methods Using polymerase chain reaction PCR technique,the gene encoding Pf12 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 pET28 a,and transfered into Escherichia coli(E.coli)strain DH5a.The recombinant plasmids were screened and identified by BamHI and XhoI digestion and PCR amplification.Using the chain termination method,Pf12 was sequenced,and the sequence of Pf12 was analysed with PCGENE software.Results The results demonstrated that the recombinant plasmid pET28 a contained the exogenous gene encoding Pf12 from isolate FCC1/HN of Plasmodium falciparum.Comparing Pf12 sequences between isolates of FCC1/HN and FMG,the results showed that there is little diffence and there may be antigenic epitopes in some domain.Conclusion These finding are important for the expression of Pf12 in vitro and development of malaria vaccine.