恶性疟原虫肝期抗原1基因3′端的克隆及序列分析  

Cloning and Sequence Analysis of 3′Region of Liver Stage Antigen 1 Gene of Plasmodium Falciparum Isolate FCC1/HN

作　　者：单志新[1] 余新炳[1] 李学荣[1] 马长玲[1] 徐劲[1] 陈守义[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》2001年第6期410-413,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】克隆并测定恶性疟原虫海南株 (FCC1/HN)肝期抗原 1基因 (LSA 1) 3′端序列 ,比较FCC1/HN株与国外分离株LSA 13′端序列的差异。【方法】应用PCR技术从FCC1/HN株基因组DNA中扩增LSA 13′端序列 ,用T A克隆法将其克隆入pMD18 T载体。阳性克隆的重组质粒经酶切及PCR扩增鉴定后 ,用双脱氧链末端终止法进行基因序列测定。应用DNASTAR软件进行不同分离株LSA 13′端序列的同源性分析。【结果】PCR扩增得到特异的FCC1/HNLSA 13′端序列。酶切及PCR鉴定获得了正确的 pT LSA 1重组质粒。测序表明 ,所克隆的LSA 13′端大小为 795bp ,编码 2 6 4个氨基酸。序列分析表明 ,我国恶性疟原虫FCC1/HN株与国外的NF5 4、T9/ 96、KEN、PNG及BRA株LSA 13′端编码的氨基酸序列只在 3个位点存在不同。【结论】克隆了恶性疟原虫FCC1/HNLSA 13′端片段。序列测定及同源性分析表明 ,FCC1/HNLSA 13′端序列与其它分离株有高度的同源性。To clone and determine the nucleotide sequence of the 3′ region of liver stage antigen gene (LSA 1) of Plasmodium falciparum isolate FCC1/HN, and to compare the differences of sequences of LSA 1 between isolate FCC1/HN and the ones abroad. The 3′ region of LSA 1 was amplified by PCR from genomic DNA of isolate FCC1/HN. Then it was cloned into pMD18 T vector by T A cloning method. The positive clones were screened and identified by agarose gel electrophoresis and endonuclease digestion and PCR technique. The correct recombinant plasmid was used as template, and the nucleotide sequence of the 3′ region of LSA 1 was determined by the dideoxy chain termination method. The DNASTAR software was used to analyze the homology of 3′ regions sequence of LSA 1 among different P.falciparum isolates. The 3′ region of LSA 1 of isolate FCC1/HN was specifically amplified,and the correct recombinant plasmid pT LSA 1 was constructed. The result of nucleotides sequencing showed that the 3′ region of LSA 1 of isolate FCC1/HN was 795 base pairs in length,encoding 264 amino acids. Only 3 sites of alternative amino acids were found in the deduced amino acids of the 3′ regions of LSA 1 among P.falciparum isolates FCC1/HN, NF54?T9/96?KEN?PNG and BRA. [Conclusion] The 3′ region of LSA 1 of P.falciparum isolates FCC1/HN was successfully cloned and sequenced. The deduced amino acids of the 3′ regions of LSA 1 of isolates FCC1/HN and other ones share quite high homology.$$$$

关 键 词：疟原虫 LSA-1 基因扩增 序列分析 恶性疟原虫 克隆 

分 类 号：R382.31[医药卫生—医学寄生虫学]