斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA的克隆和表达

Cloning and expression of Pagumogonimus skrjabini cysteine protease cDNA

目的 克隆斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA片段 ,并进行原核表达。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。将获得的cDNA片段克隆入Pin PointTMXa- 1T表达载体 ,经过筛选后 ,在大肠杆菌中进行原核表达。通过SDS -PAGE电泳和Westernblot方法鉴定表达产物 ,并检测所表达融合蛋白的免疫反应性。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 4 98bp。推导出的氨基酸序列长 16 6 ;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、组氨酸和天冬酰胺残基高度保守。原核表达中 ,对表达产物的鉴定结果和免疫反应性检测结果均显示 ,目的克隆在 33kDa处有一明显条带。结论 本实验克隆获得了斯氏狸殖吸虫囊蚴半胱氨酸蛋白酶cDNA片段 ,其序列中包含了与该酶活性有关的重要位点。原核表达获得一 33kDa的融合蛋白 ,该融合蛋白具有免疫反应性。

Aim To clone and express the cysteine protease cDNA fragment of Pagumogonimus skrjabini metacercarias.Methods The cysteine proteinase cDNA fragment was amplified by RT-PCR with degenerated primers.The production was TA-cloned into the pUCm-T vector and sequenced.DNASIS program was used to analyse the nucleotide sequence and deduce the amino acide sequence,which was aligned with the correlated parasite cysteine protease afterwards.After the ligation of PinPoint TM Xa-1 T-vector and PCR product,the clones were screened to determine the fragment orientation prior to protein expression.The expression was carried out in E.coli and analyzed on SDS-PAGE.The expressed fusion protein was identified and its immunoreactive ability was determined by Western blotting.Results A 498bp cDNA fragment was amplified by RT-PCR and sequenced.An amino acide sequence of 166 was deduced by DNASIS.Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteases and conservation of Cys,His and Asn residues that form a catalytic triad.In the identification and immunoreaction observation of the expressed fusion protein,Western blot illustrated a band of 33kDa in cells containing the PinPoint TM Xa-1 T-Vector plus the insert of interest.Conclusion The cysteine protease cDNA fragment from Pagumogonimus skrjabini metacercaria was cloned.And a fusion protein with 33kDa molecular weight is expressed.The expressed protein has immunoreactive abilitys as it reacted with immune serum.