斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段的克隆和序列分析

Cloning and sequencing of a cysteine protease cDNA fragment of adult Pagumogonimus skrjabini

目的 克隆斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段 ,并对其进行测序和序列分析。方法 利用简并引物 ,进行RT PCR ,扩增斯氏肺吸虫成虫半胱氨酸蛋白酶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扩增出了一约5 0 0bp的cDNA片段 ,对阳性克隆测序后获得其核酸序列 ,长 4 95bp。将推导的氨基酸序列作同源性分析显示 ,该序列与相关虫种半胱氨酸蛋白酶存在着较高的同源性 ,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。结论 克隆获得了斯氏肺吸虫成虫半胱氨酸蛋白酶cDNA片段 ,该片段包含了与半胱氨酸蛋白酶活性和空间结构相关的重要基因位点。

Objective To clone the cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adults, obtain the sequence and do the sequence analysis. Methods The cysteine proteinase cDNA fragment was amplified by 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 analyse the nucleotide sequence and deduce the amino acid sequence, which was aligned with the correlated parasite cysteine proteinase afterwards. Results A 495 bp cDNA fragment was amplified by RT PCR and sequenced. An amino acid sequence was deduced by DNASIS. Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteinases and conservation of Cys,His and Asn residues that form a catalytic triad. Conclusions The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adult was cloned. There are some key sites that are relevant to the function of cysteine protease in the cDNA fragment.