恶性疟原虫MESA基因的克隆和测序

目的克隆、测定恶性疟原虫海南株(FCC1／HN)成熟疟原虫感染红细胞表面抗原(MESA)基因序列，并进行序列分析。方法根据恶性疟原虫palo-alto株MESA基因已知序列，设计合成四对引物，用PCR技术从FCC1／HN株基因组DNA中扩增出4个部分序列重叠的MESA基因片段，分别克隆入pMD-18T测序载体。用双脱氧链末端终止法测定这4个基因片段的序列，拼接得到全长MESA基因序列。应用DNAstar、AnthProt软件辅助进行序列同源性比较和抗原表位区预测。结果PCR扩增得到特异的恶性疟原虫FCC1／HN株MESA基因片段，酶切及PCR鉴定获得了包含MESA基因片段的重组质粒。测序结果表明，FCC1／HN株MESA全基因编码区长4102bp，A T含量为72．11％，G C含量为27．89％，有1个内含子；编码1323个氨基酸残基，分子量为154470u。序列分析表明，FCC1／HN株与Palo-aho、D10株MESA蛋白在长度和序列组成上呈多态性，序列差异较大区域位于MESA蛋白的氨基酸重复区1、3、4、5和7。经多参数综合分析，有7个潜在的抗原表位区。结论测定、分析了恶性疟原虫FCC1／HN株MESA基因序列。FCC1／HN株MESA基因与其它分离株的MESA基因编码的氨基酸序列存在一定差异。

Cloning and DNA Sequencing of MESA Gene of Plasmodium falciparum Isolate FCC1/HN

Objective To clone and determine the nucleotide sequence of themature parasite infected erythrocyte surface antigen (MESA) gene of Plasmodium falciparum isolate FCC1/HN, and analyze the structure of MESA gene. Methods Four pairs of primers were designed according to the known sequence of MESA gene of Plasmodium falciparum isolate Palo alto. Four overlapped MESA gene fragments were amplified from genomic DNA of P. falciparum isolate FCC1/HN and cloned into pMD18 T vector by T A cloning method.The four DNA fragments were sequenced by dideoxy chain termination method to obtain the full length sequence of MESA gene. The AnthProt and DNAstar software were used to analyze the structure of MESA gene of P. falciparum isolate FCC1/HN and homology comparison of MESA antigens of P. falciparum isolates FCC1/HN, Palo alto and D10. Results Four partial MESA genes were specifically amplified from genomic DNA of P. falciparum isolate FCC1/HN and cloned into pMD18 T vector. Recombinant plasmids containing MESA gene fragments were identified by agarose gel electrophoresis, endonuclease digestion and PCR amplification. The MESA gene of isolate FCC1/HN was 4 102 base pairs in length, with 72.11 %A T and 27.89 %G C. The gene contains one intron and is encoding a protein with 1 323 amino acid residues and about 154 470u in molecular weight. The MESA antigens of P. falciparum isolate FCC1/HN, Palo alto and D10 exhibited length and amino acid residue polymorphism. The sequence diversities of MESA antigens of these P. falciparum isolates were mainly located in the repeat block 1, 3, 4, 5 and 7. The results of multiple parameters analysis showed that there were 7 potent antigenic sequential epitopes in the MESA antigen of P. falciparum isolate FCC1/HN. Conclusion The MESA gene of P. falciparum isolates FCC1/HN was cloned and DNA sequence was analyzed. Amino acid residues polymorphism in the MESA antigens of P. falciparum isolates FCC1/HN, Palo alto and D10 were found.