恶性疟原虫11.1基因3个重复片段的克隆及表达

目的　克隆和表达恶性疟原虫 (Pf) 11 1基因产物中的 3个重复片段 3R、6R和 9R。方法　利用设计的引物从培养的恶性疟原虫 3D7株的基因组DNA中扩增出 3个重复片段。PCR产物被克隆入pT7载体中以进双向测序。测序结果用GENETYX MAC软件进行分析。扩增的片段亚克隆入pET32a(+ )或pET32b(+ ) ,并由IPTG诱导在大肠杆菌BL2 1中表达重组蛋白。结果　用PCR方法成功地扩增出 3R、6R和 9R片段 ,大小分别为 5 5 2、630和44 4bp。测序结果显示 ,3D7株的Pf11 1基因比PaloAlto株的Pf11 1基因多 4个 3AA和 1个 6AA重复单元 ,两原虫株的 3R和 6R片段的同源性分别 92 8%和 95 1%。扩增出的 9R片段含有 13个 9AA重复单元。在BL2 1菌株中表达出三个重组蛋白 ,分子量分别为 45、60和 42kDa。结论　用PCR方法分别获得 3R、6R和 9R重复片段并在大肠杆菌中成功表达。 3D7株的Pf11 1基因与PaloAlto株具有高度同源性

Cloning and Expressing of the Three Repeat Fragments of Plasmodium falciparum 11 1 gene *

HT5"H]Objective To clone and express the 3R, 6R and 9R repeat fragments of Plasmodium falciparum (Pf11 1) gene. Methods Three repeat fragments from the genomic DNA of Plasmodium falciparum 3D7 strain cultivated were amplified by using the designed primers.The PCR products were cloned into the pT7 vector for bi direction sequencing.The sequencing results were analysised by GENETYX MAC. And then the amplified fragments were subcloned into pET32a( ) or pET32b( ) in order to express the recombinant proteins under the induction of IPTG in E coli BL21. Results 3R,6R and 9R fragments with sizes of 552 bp, 630 bp and 444 bp respectively were successfully amplified by PCR. The sequence analysis showed that there were 4 more 3AA units and one more 6AA unit in Pf11.1 gene of 3D7 strain as compared with Palo Alto strain. The homologies of the nucleotide sequence between the 3R fragment and the 6R fragment of the two strains were 92.8% and 95 1%,respectively. The amplified 9R fragment contains 13 9AA repeat units. The three recombinant proteins were expressed in BL21 strain with molecular weights of 45,60 and 42 kDa. Conclusion We got the 3R, 6R and 9R fragments separately by PCR and expressed them in E.coli successfully. The Pf11.1 gene of 3D7 strain is highly homologous to that of the Palo Alto strain.