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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-alto、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。
Objective To clone and determine the sequence of mature erythrocyte surface antigen (MESA) gene of Plasmodium falciparum (FCC1 / HN) in Hainan. Methods Four pairs of primers were designed and synthesized based on the known sequence of MESA gene of Plasmodium falciparum, and four overlapping sequences of MESA gene were amplified from genomic DNA of FCC1 / HN by PCR and cloned into pMD- 18T sequencing vector. The sequences of the four gene fragments were determined by dideoxy chain termination and spliced to obtain the full-length MESA gene sequence. Application DNAstar, AnthProt software to assist in the comparison of sequence homology and epitope prediction. Results The specific fragment of MESA gene of Plasmodium falciparum FCC1 / HN strain was amplified by PCR. Recombinant plasmids containing MESA gene fragment were obtained by restriction enzyme digestion and PCR. The sequencing results showed that the coding region of MESA gene of FCC1 / HN strain was 4102bp in length, 72.11% in A + T and 27.89% in G + C, encoding 1323 amino acids with a molecular weight of 154470u. Sequence analysis showed that the length and sequence composition of MESA protein of FCC1 / HN strain and Palo-alto and D10 strains were polymorphic, and the regions with large sequence differences were located in the amino acid repeat regions 1, 3, 4, 5 and 7 of MESA protein. After multi-parameter comprehensive analysis, there are 7 potential epitopes. Conclusion The MESA gene sequence of Plasmodium falciparum FCC1 / HN strain was determined and analyzed. There are some differences between the MESA gene of FCC1 / HN strain and the MESA gene of other isolates.