斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA片段的克隆和序列分析

Cloning and sequence analysis of a cysteine protease cDNA fragment from Pagumogonimus skrjabini

目的 克隆斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA片段 ,获得其序列 ,并进行序列分析。方法 利用简并引物 ,进行RT PCR ,扩增斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA片段。TA克隆装入pUCm T载体 ,进行鉴定、测序 ;利用DNASIS程序推导其所编码的氨基酸序列 ,并与相关虫种半胱氨酸蛋白酶进行氨基酸序列的同源性分析。结果 RT PCR扩增出了一 5 0 0bp左右的cDNA片段 ,对阳性克隆测序后获得其核酸序列 ,长 498bp。推导出的氨基酸序列长 166;氨基酸序列的同源性分析显示 ,该序列与相关虫种半胱氨酸蛋白酶存在着很高的同源性 ,组成半胱氨酸催化三联体的半胱氨酸、组胺酸和天冬酰胺残基高度保守。

Objective To clone the cysteine proteinase cDNA fragment from Pagumogonimus skrjabini metacercarias (PsMCP1), and do the sequence analysis. Methods The cysteine proteinase cDNA fragment was amplified by using reverse transcription polymerase chain reaction (RT PCR) with degenerate oligonucleotide primers. The product was TA cloned into the pUCm T vector and sequenced. DNASIS program was used to analyze the nucleotide sequence and deduce the amino acid sequence, which was aligned with the correlated parasite cysteine proteinase afterwards. Results A 498 bp cDNA fragment was amplified with RT-PCR. An amino acid sequence of 166 was deduced by DNASIS. Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteinases, and the Cys, His and Asn residues that formed catalytic triad were of high conservation. Conclusion The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini metacercaria is obtained in success.