杜氏利什曼原虫NH36基因的克隆及其在大肠杆菌中的表达

Cloning and expression of NH36 gene from Leishmania donovani SC10H2 strain in Escherichia coli

应用RT-PCR技术扩增并克隆杜氏利什曼原虫(Leishmania donovani)NH36编码基因,经鉴定及序列分析,构建其原核重组表达载体,经IPTG诱导表达,SDS-PAGE和Western-blotting鉴定其表达产物。结果显示,获得的NH36开放阅读框为945 bp,编码314个氨基酸残基,与GenBank上发表的国际标准株NH36编码基因序列以及氨基酸序列同源性分别为88.57%(837/945)和89.17%(280/314)。表达蛋白为36 ku,经1 mol/L IPTG诱导6 h后的表达量最高;薄层扫描显示表达的蛋白占菌体蛋白总量的57%;该蛋白可被抗杜氏利什曼原虫的多克隆抗体血清识别。

The NH36 gene was amplified from Leishrnania donovani by RT-PCR,and then cloned and sequenced. A recombinant plasmid pET-28a-Ld-NH36 was constructed and expressed by induction with IPTG. Sequencing result demonstrated that the open reading frame（（）RF） of the NH36 gene was 945 bp, encoding 314 amino acid residues, and the homologies of the nucleotide sequence and amino acid sequence to the published sequence in GenBank were 88.57 % （837/945）and 89.17 % （280/314）, respectively. SDS- PAGE indicated that the interest fusion protein was 36 ku,with peak expression 6 h after induction with 1 mol/L of IPTG. Western-blotting result showed that it was specifically recognized by polyclonal antibodies against L. donovani.