Panton-Valentine杀白细胞毒素(PVL)lukS-PV基因的克隆、原核表达及初步鉴定

Cloning and prokaryotic expression of the gene coding for LukS-PV of Panton-Valentine Leukocidin

目的构建Panton-Valentine杀白细胞毒素(PVL)lukS-PV基因表达载体,并在大肠杆菌中表达相应蛋白。方法提取PVL阳性金黄色葡萄球菌的基因,PCR方法扩增lukS-PV,A-T克隆至pGEM-T载体上,经PCR、双酶切和测序鉴定后,亚克隆到pET28a(+)表达载体上,鉴定并转化至Rosetta(DE3)plys表达宿主菌中,用IPTG诱导表达,并用SDS-PAGE电泳分析和Western blot鉴定。结果构建了lukS-PV-pGEM-T和lukS-PV-pET28a(+)重组质粒,经PCR扩增和限制性内切酶BamHI和XhoI双酶切均得到939bp的目的片段,基因测序与GENEBANK中的PVL的序列一致,宿主菌表达带HIS标签的重组LukS-PV蛋白。结论成功克隆lukS-PV基因和构建重组表达质粒,并在宿主菌中成功表达重组LukS-PV蛋白,为PVL免疫学检测方法的建立奠定基础。

Objective To construct the recombinant expression vector,and express its protein in E.coli.Methods The gene of lukS-PV was amplified by polymerase chain reaction（PCR）with the specific primers from the genomic DNA extracted in the reference strain of PVL positive Staphylococcus aureus PVL positive strain,and cloned in pGEM-T vector.Then lukS-PV gene was subcloned in the expression vector pET28a（＋）.After analyzing by PCR,restriction enzyme BamHI and XhoI,and sequencing,the recombinant plasmid of lukS-PV-pET28a（＋）was transformed into Rosetta（DE3）plysS,and induced by IPTG,followed by confirmation of Western blot.Results The length of products of PCR and test of restriction enzyme from the recombinant plasmid of lukS-PV-pGEM-T and lukS-PV-pET28a（＋）are 939bp.Sequencing result exactly consistent with that in GENEBANK.SDS-PAGE and Western blot analysis confirmed that the E.coli Rosetta（DE3）plysS with recombinant plasmid showed the high level expression of LukS-PV protein with the His tag（about 38 ku）after induction.Conclusion lukS-PV gene is cloned and the expression vector lukS-PV-pET28a（＋）is constructed successfully and the protein of LukS-PV has been expressed in Rosetta（DE3）plysS.The study provides the precondition for further study of PVL immunologic diagnosis.