恶性疟原虫Pf332基因片段的克隆及表达  

Cloning and Expression of Fragments of Pf332 Genes of Plasmodium falciparum

作　　者：单志新[1] 余新炳[1] 马长玲[1] 卞国武[1] 吴忠道[1] 徐劲[1] 

机构地区：[1]中山医科大学寄生虫学教研室,广东广州510089

出　　处：《中山医科大学学报》2002年第1期17-20,共4页Academic Journal of Sun Yat-sen University of Medical Sciences

基　　金：中山医科大学"2 11"重点学科建设课题基金资助项目 ( 9816 9);广东省自然科学基金资助项目 ( 980 0 89);教育部博士点基金资助项目 ( 93 186 )

摘　　要：【目的】构建含恶性疟原虫Pf332抗原基因片段的重组质粒 ,并检测在大肠杆菌BL2 1/DE3中的表达情况。【方法】应用PCR技术从FCC1/HN株基因组DNA中扩增Pf332基因 (Pf332 )片段 ,P332 R0、P332 R1、P332 R2 ,并分别插入pGEX 4T 1原核表达载体 ,阳性克隆的重组质粒DNA经酶切及PCR扩增鉴定。用DNAstar软件对FCC1/HN株与 3D7株Pf332的P332 R1、P332 R2片段进行同源性比较。由IPTG诱导在大肠杆菌BL2 1/DE3中表达重组蛋白。【结果】PCR扩增得到特异的FCC1/HN株Pf332片段 ,P332 R0、P332 R1、P332 R2 ,大小分别为 489、42 9和 393bp。酶切及PCR鉴定获得了正确的 pGEX P332 R0、pGEX P332 R1、pGEX P332 R2重组质粒。序列分析结果显示 ,与 3D7株相比 ,FCC1/HN株P332 R1、P332 R2片段编码多肽分别缺少 2、4个相应的重复单元。在大肠杆菌BL2 1/DE3中表达出 3个重组蛋白 ,相对分子质量分别为 46、44和 42。【结论】扩增、克隆了恶性疟原虫Pf332片段 ,P332 R0、P332 R1、P332 R2 ,并在大肠杆菌BL2 1/DE3中成功表达。FCC1/HN株与 3D7株的P332 R1、P332 R2片段编码多肽所包含的重复单元数目不同。To construct the recombinant plasmids containing the fragments of Pf332 genes and examine the expression of the Pf332 gene (Pf332) fragments in E.coli BL21/DE3. Three fragments of Pf332 genes, P332 R0, P332 R1 and P332 R2, were amplified by PCR from genomic DNA of Plasmodium falciparum (P.falciparum) isolate FCC1/HN and inserted into prokaryotic expression vector pGEX 4T 1. The positive clones were screened and identified by agarose gel electrophoresis, endonuclease digestion and PCR. The DNAstar software was used to analyze the homology of amino acids encoded by P332 R1, P332 R2 of Pf332 gene between P.falciparum isolate FCC1/HN and 3D7. The recombinant proteins were expressed under the induction of IPTG in E.coli BL21/DE3. Three fragments of Pf332 genes of P.falciparum isolate FCC1/HN, P332 R0,P332 R1 and P332 R2 with sizes of 489, 429 and 393 bp respectively were specifically amplified by PCR. The correct recombinant plasmids pGEX P332 R0, pGEX P332 R1 and pGEX P332 R2 were obtained. The results of sequence analysis showed that there were 2 and 4 absences of amino acid repeat units encoded by P332 R1 and P332 R2 of P.falciparum isolate FCC1/HN respectively,compared with those of P.falciparum isolate 3D7. The three recombinant proteins were expressed in E.coli BL21/DE3 with relative molecular mass of 46, 44 and 42. [Conclusion] Three fragments of Pf332 genes of P.falciparum isolate FCC1/HN, P332 R0, P332 R1 and P332 R2 were amplified, cloned and expressed in E.coli BL21/DE3 successfully.There were different numbers of amino acid repeat units encoded by P332 R1 and P332 R2 between P.falciparum isolate FCC1/HN and 3D7.

关 键 词：恶性疟原虫 Pf332 抗原 基因克隆 基因表达 疟原虫 

分 类 号：R382.31[医药卫生—医学寄生虫学]