华支睾吸虫蛋白酶体β2新基因的识别与克隆表达

Identification,cloning and expression ofa novel proteasome β2 gene from Clonorchis sinensis

目的识别华支睾吸虫蛋白酶体β2亚单位新基因(CsPSMB2),表达其重组蛋白,为进一步研究CsPSMB2的功能奠定基础。方法通过大规模cDNA文库随机测序,发现CsPSMB2新基因。并将其定向插入到pGEX-4T-1载体中。构建成功的载体在大肠杆菌BL21(DE3)中诱导表达,表达产物经SDS-PAGE鉴定。结果发现了编码PSMB2基因的一个新成员CsPSMB2,CsPSMB2全长708个核苷酸,编码一个201个氨基酸残基的蛋白,理论分子量为22.5kD,预测的蛋白与人的PSMB2蛋白同源性为58%并且含有一个蛋白酶体结构域,成功登录GeneBank,登录号为AY849972。构建成功重组质粒pGEX-4T-1-CsPSMB2,经IPTG诱导后表达出PSMB2的GST融合蛋白。结论发现华支睾吸虫新基因CsPSMB2,成功构建了pGEX-4T-1-CsPSMB2重组表达载体并表达出了CsPSMB2的GST融合蛋白。为进一步研究CsPSMB2在华支睾吸虫中的功能和可能的应用研究奠定基础。

This study was attempted to isolate a novel proteasome β2 gene （CsPSMB2） from eDNA library of Clonorchis sinensis adult worm, and the eDNAs were identified by large-scale sequencing of the C. sinensis adult worms. A pair of oligonueleotide primers was designed according to the coding sequence of CsPSMB2. The coding region of this gene was amplified by PCR and then cloned into the plasmid vector pGEX-4T-1, and the recombinant plasmid identified by PCR and endonuelease digestion was then transformed to BL21（DE3） cells. After the indution with IPTG, the fusion protein was expressed and identified by SDS-PAGE. The experimental results showed that a novel CsPSMB2 gene was identified from eDNA library of C. sinensis adult worm and the 708 bp eDNA encoded a putative protein of 201 amino acids with a predicted molecular mass of 22.5 kDa. The putative amino acid contained a single proteasome domain with 58% homologue with human PSMB2 protein. A recombinant plasmid pGEX-4T-1-CsPSMB2 was constructed with the coding number of AY849972 and it could express the GST fusion protein after IPTG induction. It suggests that a novel CsPSMB2 gene is identified from eDNA library of C. sinensis adult worm.