本文選題：肺炎鏈球菌 + 肺炎鏈球菌表面粘附素A　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2007年碩士論文

【摘要】： 肺炎鏈球菌(Streptococcus pneumoniae，Sp)有90個血清型，其中近30個血清型對人有致病性，是細菌性肺炎的主要病原體之一。當Sp從感染的原發(fā)部位播散到循環(huán)系統(tǒng)、中樞神經(jīng)系統(tǒng)或其它正常情況下無菌部位時，會引起菌血癥、腦膜炎等侵襲性疾病。 肺炎鏈球菌表面粘附素A(pneumococcal surface adhesin A，PsaA)是各型Sp共有的一種遺傳保守、種特異性的表面結合脂蛋白，在細菌侵襲呼吸道粘膜過程中起關鍵的粘附作用，因此與細菌的侵襲性和毒力有關；PsaA具有很好的抗原性，機體感染Sp后會產(chǎn)生抗PsaA特異性抗體。PsaA編碼開放閱讀框(Open reading frame，ORF)為930 bp，分子量約為37kD，成熟PsaA空間結構為1個α螺旋連接2個(β／α)_4結構域。 本研究采用聚合酶鏈反應(polymerse chain reaction，PCR)擴增Sp psaA基因，并插入原核質(zhì)粒pET-32a，獲得重組質(zhì)粒pET32a-psaA，其中以伯氏疏螺旋體外膜蛋白OspA信號肽基因序列(51bp)替換基因psaA自身信號肽基因序列。測序表明，目的基因以正確的開放讀碼框插入表達載體。將重組質(zhì)粒pET32a-psaA通過轉化進入重組表達載體大腸桿菌BL21plysS中，經(jīng)IPTG誘導，獲得rPsaA高效表達，表達量約為菌體蛋白的60％。氨基酸序列分析表明重組PsaA(recombinant PsaA，rPsaA)與天然PsaA的同源性為99.3％。表達產(chǎn)物的SDS-PAGE分析表明，rPsaA蛋白分子量約為52kD，，以可溶性表達為主。Western blot分析表明，rPsaA蛋白可與Sp免疫兔血清發(fā)生反應，說明rPsaA蛋白具有較好的抗原性。rPsaA蛋白以50％飽和硫酸銨純化，其純度可達80％以上。 本研究以rPsaA蛋白作為捕獲抗原制備免疫膠體金試劑。初步檢測顯示，該試劑檢測Sp免疫兔血清呈陽性，檢測正常兔血清和生理鹽水呈陰性；應用Sp免疫膠體金試劑檢測72份健康人血清標本，其中70份檢測陰性，2份檢測弱陽性(稀釋度均為1：100)，說明試劑特異性較好，rPsaA蛋白有望用于Sp肺炎診斷。
[Abstract]:Streptococcus pneumoniae spp has 90 serotypes, of which nearly 30 serotypes are pathogenic to human beings and are one of the main pathogens of bacterial pneumonia.When Sp spreads from the primary site of infection to the circulatory system, central nervous system or other normal sterile parts, it can cause invasive diseases such as bacteremia, meningitis, and so on.Streptococcus pneumoniae surface adhesion hormone (A(pneumococcal surface adhesin A PsaA) is a conserved and specific surface binding lipoprotein, which plays a key role in bacterial invasion of respiratory mucosa.Therefore, PsaA has good antigenicity related to the invasiveness and virulence of bacteria.The specific antibody against PsaA. PsaA encoding open reading frame is 930 BP, molecular weight is about 37 kD. the mature PsaA spatial structure is a 偽 helix connected with two (尾 / 偽 4) domains.In this study, the Sp psaA gene was amplified by polymerase chain reaction polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid pET-32a. The recombinant plasmid pET32a-psaA was obtained, in which the gene sequence of psaA signal peptide gene was replaced by the sequence of OspA signal peptide gene of Borrelia burgdorferi outer membrane protein (pET32a-psaA).Sequencing showed that the target gene was inserted into the expression vector with the correct open reading frame.The recombinant plasmid pET32a-psaA was transformed into Escherichia coli BL21plysS and induced by IPTG to obtain the high expression of rPsaA. The expression amount was about 60% of the bacterial protein.Amino acid sequence analysis showed that the homology between recombinant PsaA(recombinant PsaA rPsaA and natural PsaA was 99.3%.SDS-PAGE analysis showed that the molecular weight of rPsaA protein was about 52 kD. Western blot analysis showed that rPsaA protein could react with the serum of rabbits immunized with Sp, indicating that rPsaA protein had good antigenicity. The protein was purified with 50% saturated ammonium sulfate.Its purity can reach more than 80%.In this study, rPsaA protein was used as capture antigen to prepare immune colloidal gold reagent.The results showed that Sp immunized rabbit serum was positive, normal rabbit serum and normal saline were negative, and Sp immunocolloidal gold reagent was used to detect 72 healthy human serum samples.Of them, 70 were negative and 2 were weakly positive (dilution was 1: 100), which indicated that the specificity of the reagent was better and the rPsaA protein could be used in the diagnosis of SP pneumonia.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R378

【引證文獻】

相關碩士學位論文 前1條

1 趙海;牛分枝桿菌主要分泌性蛋白70和脂蛋白83的克隆表達及抗原性研究[D];中國人民解放軍軍事醫(yī)學科學院;2010年

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