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目的比对分析微小隐孢子虫南京(NJ)株亲环素-RNA相互作用蛋白(CRIP)与其他隐孢子虫株CRIP基因序列的差异。方法根据GenBank微小隐孢子虫IowaⅡ株CRIP基因序列设计并合成2对引物,应用巢式聚合酶链反应(PCR)技术从微小隐孢子虫NJ株基因组DNA中扩增CRIP基因,并将其克隆到pMD18-T载体上,将重组质粒pMD18-T-CpCRIP进行PCR和双酶切鉴定后测序,应用生物信息学方法分析微小隐孢子虫NJ株CRIP基因与其他种属的CRIP基因序列同源性。结果巢式PCR扩增获得的特异性CRIP基因序列,经PCR及双酶切鉴定确为pMD18-T-CpCRIP重组质粒;测序结果显示,该序列有119 bp,微小隐孢子虫NJ株CRIP基因全长为909bp,编码302个氨基酸;测序结果和同源性分析显示,中国微小隐孢子虫NJ株CRIP基因序列与国外的微小隐孢子虫Iowa II株同源性为98%;该隐孢子虫NJ株CRIP基因序列获GenBank登录号JQ396883。结论微小隐孢子虫NJ株CRIP基因与其他微小隐孢子虫株存在基因变异。
Objective To compare and analyze the differences of the CRIP gene between CRIP gene and other Cryptosporidium strains in Nanjing (NJ) isolates. Methods Two pairs of primers were designed and synthesized according to the sequence of CRIP gene of GenBank Iowa strain. The CRIP gene was amplified from genomic DNA of Cryptosporidium parvum NJ using nested polymerase chain reaction (PCR) and cloned into On the pMD18-T vector, the recombinant plasmid pMD18-T-CpCRIP was identified by PCR and double restriction enzyme digestion. The bioinformatics methods were used to analyze the sequence homology of the CRIP gene between Cryptosporidium parvum NJ strains and other species. Results The specific CRIP gene sequence obtained by nested PCR amplification was identified by PCR and double enzyme digestion as pMD18-T-CpCRIP recombinant plasmid. The sequencing result showed that the sequence of CRIP gene was 119 bp. The CRIP gene of Cryptosporidium parvum NJ Length of 909bp, encoding 302 amino acids; sequencing results and homology analysis showed that China Cryptosporidium NJ CRIP gene sequence and foreign Cryptosporidium parvum Iowa II homology was 98%; Cryptosporidium NJ The strain CRIP gene sequence was obtained from GenBank accession number JQ396883. Conclusion There is genetic variation between CRIP gene of Cryptosporidium parvum and other Cryptosporidium parvum strains.