本文選題：金黃色葡萄球菌　切入點：Panton-Valentine殺白細胞毒素　出處：《安徽醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:克隆金黃色葡萄球菌的Panton-Valentine殺白細胞毒素(PVL)基因,構(gòu)建原核表達載體pET28a/lukS-PV和pET28a/lukF-PV,表達LukS-PV和LukF-PV重組蛋白,利用此重組蛋白免疫新西蘭白兔制備多抗血清。方法:根據(jù)PUBMED公布的PVL序列設(shè)計引物,并引入BamH I和Xho I酶切位點。應(yīng)用PCR擴增出lukS-PV和lukF-PV全長基因片斷。將目的基因lukS-PV和lukF-PV分別插入克隆載體pGEM-T,提取重組質(zhì)粒,BamH I和Xho I雙酶切獲得目的基因,插入原核表達質(zhì)粒pET28a中,重組子雙酶切、PCR和測序鑒定,轉(zhuǎn)化大腸桿菌Rosetta(DE3)plysS并以IPTG誘導(dǎo)表達。親和層析法純化重組蛋白,SDS-PAGE和Western blotting鑒定重組蛋白,并以此免疫新西蘭白兔,收集抗血清,ELISA測定滴度,Western blotting鑒定免疫活性。結(jié)果:PCR從PVL陽性的金黃色葡萄球菌銅23株中擴增出939bp的lukS-PV和978bp的lukF-PV目標(biāo)基因片斷。并成功克隆入pET28a。pET28a/lukS-PV和pET28a/lukF-PV經(jīng)BamH I和Xho I雙酶切,獲得與目標(biāo)基因大小一致的基因片斷,測序結(jié)果與GenBank比對LukS-PV和LukF-PV同源性均達99%。攜帶有pET28a/lukS-PV和pET28a/lukF-PV。重組質(zhì)粒的宿主菌Rosetta(DE3)plysS分別經(jīng)誘導(dǎo)高效表達上述兩種蛋白質(zhì)。SDS-PAGE檢測其分子量:rLukS-PV和rLukF-PV的分子量分別約為38kDa和39kDa,二者與預(yù)期的分子量相符。經(jīng)Ni親和層析法純化了rLukF-PV蛋白,免疫新西蘭白兔,獲得ELISA滴度為10-3的多價特異性抗血清,Western blotting顯示,多價血清不僅能和其相應(yīng)的重組蛋白而且和PVL陽性的金黃色葡萄球菌發(fā)生特異性免疫反應(yīng)。結(jié)論:成功的從PVL陽性的金黃色葡萄球菌基因組中獲取了lukS-PV和lukF-PV基因,構(gòu)建了pET28a/lukS-PV和pET28a/lukF-PV重組質(zhì)粒,并表達了相應(yīng)重組蛋白,進而用rLukF-PV免疫新西蘭白兔獲得了多克隆抗血清。本研究為建立免疫法快速檢測PVL陽性金黃色葡萄球菌奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【參考文獻】

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