华支睾吸虫蛋白酶体β1新基因克隆与表达
Molecular cloning and expression of a novel proteasome β1 gene from Clonorchis sinensis
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摘要:目的 通过筛选华支睾吸虫cDNA文库发现蛋白酶体β1(CsPSMB1)新基因, 表达其重组蛋白, 为进一步研究CsPSMB1的功能及应用提供基础数据。方法 将插入于pBluscripetⅡSK(+)载体中的cDNA随机测序, 发现CsPSMB1新基因。根据pGEX-4T-1的多克隆位点及CsPSMB1编码区序列设计引物, 进行PCR扩增, 双酶切后连接到pGEX-4T-1载体中。重组表达质粒pGEX-4T-1-CsPSMB1经PCR、双酶切及测序鉴定后, 转化大肠埃希菌BL21(DE3), 异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达, 表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。结果 发现了编码PSMB1基因的一个新成员CsPSMB1, CsPSMB1全长749个核苷酸, 编码一个217个氨基酸残基的蛋白, 理论分子量为24kD, 预测的蛋白与人的PSMB1蛋白同源性为50%并且含有一个蛋白酶体结构域。序列已经成功登录GeneBank, 登录号为AY849971。构建成功重组质粒pGEX-4T-1-CsPSMB1, 经IPTG诱导后表达出PSMB1的GST融合蛋白。结论 发现了华支睾吸虫新基因CsPSMB1, 成功构建pGEX-4T-1-CsPSMB1重组表达载体并表达出了CsPSMB1的GST融合蛋白。Abstract:Objective To explore the proteasome B1 gene(CsPSMB1)from Clonor chis sinensis.To provide the functional study foundation of the CsPSMB1.Methods From a cDNA library of Clonorchis sinensis adult worm, the cDNA inserts in pBluescriptⅡSK(+) were identified by large-scale sequencing and a novel CsPSMB1 gene were identified.A pair specific oligonucleotide primers were designed and syntheses according to the coding sequence of CsPSMB1.The coding region of CsPSMB1 was amplified by PCR and then cloned into the vector of pGEX-4T-1.The recombinant plamid was identified by PCR and endonuclease digestion then transformed into BL21(DE3).The fusion protein was expression after IPTG induction for 3 hours and identified by SDS-PAGE.Results A novel CsPSMB1 gene was identified from Clonorchis sinensis adult worm cDNA library.The 749 bp cDNA encoded a putative protein of 217 amino acids with a predicted molecular mass of 24kD.The putative protein contained a single proteasome domain and founded to be 50% identical to the PSMB1 of homo sapiens.A recombinant plamid pGEX-4T-1-CsPSMB1 was constructed and expressed the GST fusion protein by IPTG induce.Conclusion A novel CsPSMB1 gene was identified from Clonorchis sinensis adult worm cDNA library.A recombinant plamid pGEX-4T-1-CsPSMB1 was constructed and expressed the GST fusion protein by IPTG induce.
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