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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。
Objective To clone the coding region of Pfmdr1 and Cg1 genes of Plasmodium falciparum FCC1 / HN strain (CQS) into sequencing vector, and to determine the sequence of Pfmdr1 and Cg1 gene for further study on its relationship with Plasmodium resistance. Methods The full-length Pfmdr1 gene was amplified from genomic DNA of Plasmodium falciparum FCC1 / HN strain in three fragments by PCR amplification. The full-length coding sequence of Cg1 gene was amplified by two fragments. After purification and recovery of the amplified product, the T-A gene was cloned into the sequencing vector pMD18-T and transformed into E. coli JM109. The positive clones were screened and identified by double enzyme digestion and PCR amplification. Positive recombinant plasmids were obtained, The DNA assay was performed using the Sanger dideoxy chain termination method. Results The Pfmdr1 gene of CQSFCC1 / HN strain was highly homologous to the CQSFc2 7 strain. It was 4 2 48 bp in length and encoded 14 15 amino acids. The full-length Cg1 gene was 2 82 0 bp encoding 939 amino acids with a tandem α repeat sequence. Conclusion The determination of the coding sequence of Pfmdr1 and Cg1 genes in Plasmodium falciparum FCC1 / HN (CQS) strain will provide a basis for further study on the above genes of resistant strains and their relationship with Plasmodium resistance.