恶性疟原虫FCC1/HN(CQS)株Pfmdr1、Cg1基因T-A克隆及序列分析

T-A cloning and sequence analysis of the gene Pfmdr1 and Cg1 ofPlasmodium falciparum isolate FCC1/HN(CQS)

目的 将恶性疟原虫FCC1/HN株 (CQS)Pfmdr1和Cg1全基因编码区克隆入测序载体 ,测定其序列 ,为以后研究其与疟原虫耐药性的关系奠定基础。方法 利用PCR扩增技术 ,分 3个片段从恶性疟原虫FCC1/HN株基因组DNA中 ,特异扩增Pfmdr1全基因编码序列 ;分 2个片段特异扩增Cg1全基因编码序列。扩增产物经纯化回收后 ,T -A克隆入测序载体 pMD18-T ,转化大肠杆菌 (E coli)JM 10 9,筛选阳性克隆 ,并进行双酶切及PCR扩增鉴定 ,获得阳性重组质粒 ,用Sanger双脱氧链终止法进行DNA测定。结果 CQSFCC1/HN株Pfmdr1基因序列与CQSFc2 7株同源性高 ,全长 4 2 4 8bp ,编码 14 15个氨基酸 ;测得Cg1基因全长为 2 82 0bp ,编码 939个氨基酸 ,存在串联α重复序列。 结论 恶性疟原虫FCC1/HN(CQS)株Pfmdr1和Cg1全基因编码序列的测定 ,为以后研究耐药性虫株的上述基因以及它们与疟原虫耐药性的关系奠定基础。

Aim Sequence the Pfmdr1 and Cg1 of Plasmodium falciparum isolate FCC1/HN(CQS)for its structure and functional studies Methods According to the published nucleotide sequence of the Pfmdr1 and Cg1,primers were designed Using polymerase chain reaction(PCR)technique,the gene of Pfmdr1 and Cg1 were specifically amplified from the genomic DNA of P falciparum isolate FCC1/HN(CQS) The PCR products were puried and cloned into plasmid pMD18 T by T A cloning method,transformed into Escherichia coli(E coli)strain JM109 The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification Results DNA sequence of the Pfmdr1 and Cg1 gene were 4,248bp and 2,820 bp respectively The predicted protein sequences were highly conserved Conclusion These findings are important for the studies of two genes which may be linked to chloroquine resistance in Plasmodium falciparum