金黄色葡萄球菌Panton-Valentine杀白细胞毒素（PVL）基因的克隆表达及其抗体的制备

发布时间：2018-03-06 07:10

本文选题：金黄色葡萄球菌　切入点：Panton-Valentine杀白细胞毒素　出处：《安徽医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:克隆金黄色葡萄球菌的Panton-Valentine杀白细胞毒素(PVL)基因,构建原核表达载体pET28a/lukS-PV和pET28a/lukF-PV,表达LukS-PV和LukF-PV重组蛋白,利用此重组蛋白免疫新西兰白兔制备多抗血清。方法:根据PUBMED公布的PVL序列设计引物,并引入BamH I和Xho I酶切位点。应用PCR扩增出lukS-PV和lukF-PV全长基因片断。将目的基因lukS-PV和lukF-PV分别插入克隆载体pGEM-T,提取重组质粒,BamH I和Xho I双酶切获得目的基因,插入原核表达质粒pET28a中,重组子双酶切、PCR和测序鉴定,转化大肠杆菌Rosetta(DE3)plysS并以IPTG诱导表达。亲和层析法纯化重组蛋白,SDS-PAGE和Western blotting鉴定重组蛋白,并以此免疫新西兰白兔,收集抗血清,ELISA测定滴度,Western blotting鉴定免疫活性。结果:PCR从PVL阳性的金黄色葡萄球菌铜23株中扩增出939bp的lukS-PV和978bp的lukF-PV目标基因片断。并成功克隆入pET28a。pET28a/lukS-PV和pET28a/lukF-PV经BamH I和Xho I双酶切,获得与目标基因大小一致的基因片断,测序结果与GenBank比对LukS-PV和LukF-PV同源性均达99%。携带有pET28a/lukS-PV和pET28a/lukF-PV。重组质粒的宿主菌Rosetta(DE3)plysS分别经诱导高效表达上述两种蛋白质。SDS-PAGE检测其分子量:rLukS-PV和rLukF-PV的分子量分别约为38kDa和39kDa,二者与预期的分子量相符。经Ni亲和层析法纯化了rLukF-PV蛋白,免疫新西兰白兔,获得ELISA滴度为10-3的多价特异性抗血清,Western blotting显示,多价血清不仅能和其相应的重组蛋白而且和PVL阳性的金黄色葡萄球菌发生特异性免疫反应。结论:成功的从PVL阳性的金黄色葡萄球菌基因组中获取了lukS-PV和lukF-PV基因,构建了pET28a/lukS-PV和pET28a/lukF-PV重组质粒,并表达了相应重组蛋白,进而用rLukF-PV免疫新西兰白兔获得了多克隆抗血清。本研究为建立免疫法快速检测PVL阳性金黄色葡萄球菌奠定基础。
[Abstract]:Objective: to clone staphylococcus aureus (Staphylococcus aureus) Panton-Valentine leukocytotoxin (PVL) gene, construct prokaryotic expression vectors pET28a/lukS-PV and pET28a / lukF-PVand express LukS-PV and LukF-PV recombinant proteins. The recombinant protein was used to immunize New Zealand white rabbits to prepare polyantibodies. Methods: primers were designed according to the PVL sequence published by PUBMED. The full-length lukS-PV and lukF-PV gene fragments were amplified by PCR. The target gene lukS-PV and lukF-PV were inserted into the clone vector pGEM-Trespectively, and the recombinant plasmids were digested with BamH I and Xho I to obtain the target gene. The recombinant plasmid was inserted into the prokaryotic expression plasmid pET28a. The recombinant plasmid was digested by PCR and sequenced. The recombinant protein was transformed into Escherichia coli Rosetta(DE3)plysS and expressed by IPTG. The recombinant protein was purified by affinity chromatography and identified by Western blotting, and the recombinant protein was immunized with the recombinant protein. Results 939bp lukS-PV and 978bp lukF-PV target gene fragments were amplified from 23 PVL positive strains of S.aureus by blotting. PET28a.pET28a/lukS-PV and pET28a/lukF-PV were cloned into pET28a.pET28a/lukS-PV and pET28a/lukF-PV by BamH I and Xho I double enzyme digestion. Get a gene fragment that is the same size as the target gene, The homology of LukS-PV and LukF-PV reached 99k.The recombinant plasmid carrying pET28a/lukS-PV and pET28a / lukF-PV.Recombinant plasmid Rosetta(DE3)plysS was induced to express the two proteins. SDS-PAGE showed that the molecular weight of Rosetta(DE3)plysS and rLukF-PV were about 38kDa and 39kDarespectively. RLukF-PV protein was purified by Ni affinity chromatography. New Zealand white rabbits were immunized with polyvalent antiserum (ELISA titer 10-3). The polyvalent serum could not only react specifically with the corresponding recombinant protein but also with the PVL positive Staphylococcus aureus. Conclusion: the lukS-PV and lukF-PV genes were successfully obtained from the PVL positive Staphylococcus aureus genome. The recombinant plasmids of pET28a/lukS-PV and pET28a/lukF-PV were constructed and the corresponding recombinant proteins were expressed. The polyclonal antiserum was obtained by immunizing New Zealand white rabbits with rLukF-PV. This study laid a foundation for the rapid detection of PVL positive Staphylococcus aureus by immunoassay.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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