肺炎链球菌表面粘附素A（PsaA）基因的克隆表达及初步鉴定

发布时间：2018-04-12 06:03

本文选题：肺炎链球菌 + 肺炎链球菌表面粘附素A　；参考：《中国人民解放军军事医学科学院》2007年硕士论文

【摘要】： 肺炎链球菌(Streptococcus pneumoniae，Sp)有90个血清型，其中近30个血清型对人有致病性，是细菌性肺炎的主要病原体之一。当Sp从感染的原发部位播散到循环系统、中枢神经系统或其它正常情况下无菌部位时，会引起菌血症、脑膜炎等侵袭性疾病。肺炎链球菌表面粘附素A(pneumococcal surface adhesin A，PsaA)是各型Sp共有的一种遗传保守、种特异性的表面结合脂蛋白，在细菌侵袭呼吸道粘膜过程中起关键的粘附作用，因此与细菌的侵袭性和毒力有关；PsaA具有很好的抗原性，机体感染Sp后会产生抗PsaA特异性抗体。PsaA编码开放阅读框(Open reading frame，ORF)为930 bp，分子量约为37kD，成熟PsaA空间结构为1个α螺旋连接2个(β／α)_4结构域。本研究采用聚合酶链反应(polymerse chain reaction，PCR)扩增Sp psaA基因，并插入原核质粒pET-32a，获得重组质粒pET32a-psaA，其中以伯氏疏螺旋体外膜蛋白OspA信号肽基因序列(51bp)替换基因psaA自身信号肽基因序列。测序表明，目的基因以正确的开放读码框插入表达载体。将重组质粒pET32a-psaA通过转化进入重组表达载体大肠杆菌BL21plysS中，经IPTG诱导，获得rPsaA高效表达，表达量约为菌体蛋白的60％。氨基酸序列分析表明重组PsaA(recombinant PsaA，rPsaA)与天然PsaA的同源性为99.3％。表达产物的SDS-PAGE分析表明，rPsaA蛋白分子量约为52kD，，以可溶性表达为主。Western blot分析表明，rPsaA蛋白可与Sp免疫兔血清发生反应，说明rPsaA蛋白具有较好的抗原性。rPsaA蛋白以50％饱和硫酸铵纯化，其纯度可达80％以上。本研究以rPsaA蛋白作为捕获抗原制备免疫胶体金试剂。初步检测显示，该试剂检测Sp免疫兔血清呈阳性，检测正常兔血清和生理盐水呈阴性；应用Sp免疫胶体金试剂检测72份健康人血清标本，其中70份检测阴性，2份检测弱阳性(稀释度均为1：100)，说明试剂特异性较好，rPsaA蛋白有望用于Sp肺炎诊断。
[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 伪 helix connected with two (尾 / 伪 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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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