肺炎支原体多抗原表位表达载体的构建与鉴定

发布时间：2018-06-16 03:09

本文选题：肺炎支原体 + 蛋白抗原　；参考：《中国医科大学》2010年硕士论文

【摘要】： 前言肺炎支原体(Mycoplasma pneumoniae,Mp)是人类原发性非典型肺炎的病原体,无细胞壁,其引起感染的治疗与其它细菌和病毒感染有所不同,因此,Mp感染诊断意义重大。Mp细胞表面的粘附蛋白(如P1,P116等)能与宿主细胞受体结合,为Mp致病的重要因素,也是引起机体免疫反应的主要免疫原,应用重组蛋白抗原检测临床标本,有较高的特异性和敏感性。国内外已有人成功构建了Mp P1蛋白的表达载体。为了增加重组蛋白的抗原表位,提高诊断的敏感性,本研究经PCR点突变技术(TGA→TGG)获取Mp116蛋白目的基因,构建克隆载体,并与原实验室构建的PGEX-6P-1-P1表达载体重组,构建肺炎支原体多抗原表位表达载体,从克隆株中提取纯化重组蛋白,经Western blotting鉴定融合蛋白的免疫反应性,为研制更有效的肺炎支原体基因工程诊断试剂奠定基础。方法 1.Mp DNA的制备按本实验室常规方法进行。 2、PCR点突变扩增Mp 116目的基因根据GenBank提供的Mp 116蛋白基因(2467—3051)序列,通过引物分析软件Primer5.0和OLIGO6设计引物,上游引物序列：5'-TTTTGGATCCTCAAA GATAACATCCAAGTG-3'(BamI),下游引物序列：5'-ATATGAATTCTTGAAGCC CATGTCAGTAAAG-3'(EcoRI),突变引物序列：5'-GACCTTTGGTTGTTTAAGA TTTGGCCTAAGTTC-3'.PCR扩增突变片段,加20μM的突变引物和下游引物以及100ng模板,反应总体积50μl,95℃5min×1；95℃30s,55℃30s,72℃40s×30；72℃10min×1。回收突变片段,以此做引物扩增目的基因,加入20μM上游引物和100ng模板,95℃5min×1；95℃1min,65℃1min,72℃1min×32；72℃10min×1。1.0%琼脂糖凝胶电泳鉴定。 3、Mp克隆载体PMD-T-116的构建与鉴定将回收的目的基因和PMD-T载体连接,转入E.coli JM109。通过氨苄青霉素平板筛选阳性克隆株。质粒提取试剂盒抽提阳性克隆株的重组质粒,PCR扩增及酶切后电泳鉴定。插入的基因序列由南京金思特科技公司测定,并与GenBank提供的FH株肺炎支原体P116核苷酸序列进行同源性比较。 4、Mp多抗原表位表达载体P1-P116的构建与鉴定从克隆株中提取纯化重组质粒,BamHI、EcoRI双酶切后进行琼脂糖凝胶电泳,回收目的片段,与BamHI、EcoRI双酶切的PGEX-6P-1-P1表达载体(P1表达载体为EcoRI、XhoI双酶切构建)连接,转入E.coli JM109。氨苄青霉素平板筛选转化子,提取纯化重组质粒,质粒酶切图谱和PCR扩增鉴定。 5、重组蛋白基因表达的检测与鉴定重组质粒P1-P116转入E.coli BL21中,将阳性克隆株培养菌液接种于含氨苄青霉素(100μg/ml)的LB液体培养基中,37℃振荡培养过夜。离心收集菌体沉淀,转接至200ml氨苄青霉素LB中,37℃振荡培养30 min,加入IPTG至终浓度0.5mmol/L,诱导表达2h。离心收集菌体沉淀。冰裕超声破碎,离心后取上清用Glutathione Sepharose 4B纯化GST-P1-P116融合蛋白。SDS-PAGE分析表达产物的相对分子量,免疫印迹实验鉴定其免疫反应性。结果 1、P116目的基因片段的扩增与鉴定 PCR扩增的突变引物片段为176bp,扩增的目的基因片段为597bp,经1.0%琼脂糖凝胶电泳鉴定,扩增产物与理论值大小基本相符。 2、PMD-T-116重组质粒鉴定以PMD-T-116为模板,PCR扩增出单一条带,长度与预期一致,PMD-T-116用BamHI、EcoRI双酶切,产生2.7kb和0.6kb两个片段。插入基因片段与已知的P116核苷酸序列进行比较,除突变碱基外,其余完全一致。 3、Mp表达载体的构建与鉴定以重组质粒为模板,PCR扩增出单一条带,长度与预期一致。重组质粒用BamHI、EcoRI双酶切产生0.6kb和5.8kb两个片段,用EcoRI、Xhol双酶切产生0.9kb和5.5kb两个片段,用BamHI、Xhol双酶切产生1.5kb和4.9kb两个片段。 4、重组蛋白抗原性检测经诱导后表达相对分子质量(Mr)为81KDa的融合蛋白,与预期的Mr大小一致。Western blotting结果显示,兔多价抗血清与纯化的融合蛋白在81KDa处形成了一条特异的抗原—抗体反应条带。结论 1、本实验经PCR点突变(TGA→TGG)获取Mp 116蛋白羧基端目的基因片段。 2、构建了P1-P116双蛋白多抗原表位的表达载体,转入大肠杆菌内,可稳定地表达融合蛋白。 3、Western blotting证明表达的81KDa的融合蛋白具有免疫反应性。
[Abstract]:Preface
Mycoplasma pneumoniae (Mp), a pathogen of human primary atypical pneumonia, is the pathogen of primary atypical pneumonia and has no cell wall. The treatment of infection is different from other bacterial and viral infection. Therefore, Mp infection is important for the diagnosis of the adhesion proteins on the surface of.Mp cells (such as P1, P116, etc.), which can be combined with the host cell receptor, which is an important cause for the pathogenesis of Mp. It is also the main immunogen that causes the immune response of the body. The recombinant protein antigen is used to detect the clinical specimens with high specificity and sensitivity. The expression vector of Mp P1 protein has been successfully constructed at home and abroad. In order to increase the epitopes of the recombinant protein and improve the sensitivity of the diagnosis, this study has been obtained by PCR point mutation (TGA to TGG). In order to develop a more effective gene engineering of Mycoplasma pneumoniae, the target gene of Mp116 protein was obtained, and the cloning vector was constructed and the recombinant PGEX-6P-1-P1 expression vector was constructed in the original laboratory to construct a multi antigen epitope expression vector of Mycoplasma pneumoniae. The recombinant protein was extracted and purified from the clone, and the immunoreactivity of the fusion protein was identified by Western blotting. The diagnostic reagent lays the foundation.
Method
Preparation of 1.Mp DNA
It is carried out in the routine method of this laboratory.
2, PCR point mutation amplification of Mp 116 gene
According to the Mp 116 protein gene (2467 - 3051) sequence provided by GenBank, primers were designed by the primer analysis software Primer5.0 and OLIGO6. The sequence of upstream primers: 5'-TTTTGGATCCTCAAA GATAACATCCAAGTG-3'(BamI), the sequence of downstream primers: 5'-ATATGAATTCTTGAAGCC CATGTCAGTAAAG-3' (EcoRI), mutation primer sequence: 5'-GACCTTTGGTTGTTTAAGA TTTGGCCT AAGTTC-3'.PCR amplified mutant fragments, adding 20 mu M mutation primers and downstream primers and 100ng templates, the total volume was 50 mu L, 95 C 5min x 1, 95 C 30s, 55 C 30s, 72 C 40s x 30, 72 C 10min x 1. recovery fragment, which was amplified by primers and 100ng templates, 95, 5min * 1, 95 degrees 5min * 1; 95 degrees C The temperature was 72 1min 10min 32 1.1.0% and identified by agarose gel electrophoresis.
3, construction and identification of Mp cloning vector PMD-T-116
The reclaimed target gene and PMD-T vector were connected to E.coli JM109. to screen positive clones through ampicillin plate. Plasmid extraction kits were used to extract the recombinant plasmid of positive clones, PCR amplification and electrophoresis identification after enzyme digestion. The inserted gene sequence was determined by Nanjing Jin steet technology company, and the FH strain pneumonia branch provided with GenBank. The homology of the P116 nucleotide sequence of the plasma was compared.
4, construction and identification of Mp multi epitope expression vector P1-P116.
The recombinant plasmid was extracted and purified from the clones. BamHI and EcoRI were cut into agarose gel electrophoresis to recover the target fragments. The PGEX-6P-1-P1 expression vector (P1 expression vector for EcoRI, XhoI double enzyme cut construction) was connected with BamHI, EcoRI double enzyme, and transformed into the E.coli JM109. ampicillin plate to screen the transformant and extract the recombinant plasmid and plasmid. Enzyme digestion and PCR amplification were identified.
5, detection and identification of recombinant protein gene expression
The recombinant plasmid P1-P116 was transferred into E.coli BL21 to inoculate the positive clone culture liquid in the LB liquid medium containing ampicillin (100 u g/ml) and oscillate at 37 degrees for the night. Centrifugation was carried out to collect bacteria to precipitate and transfer to 200ml ampicillin LB. 30 min was cultured at 37 degrees C, and IPTG to the final concentration 0.5mmol/L was added to induce expression 2h. centrifugation collection. The bacteria were precipitated. Ice margin was broken by ultrasound. After centrifugation, Glutathione Sepharose 4B was used to purify the GST-P1-P116 fusion protein.SDS-PAGE to analyze the relative molecular weight of the expression products, and the immunoblotting test was used to identify the immune response.
Result
1, amplification and identification of P116 target gene fragment
The amplified mutation primer fragment of PCR was 176bp, and the amplified target gene fragment was 597bp. The amplified product was identified by 1% agarose gel electrophoresis. The amplified product was basically consistent with the theoretical value.
2, PMD-T-116 recombinant plasmid identification
Using PMD-T-116 as a template, PCR amplified a single band with the same length as expected. PMD-T-116 used BamHI, EcoRI double enzyme to produce two fragments of 2.7kb and 0.6kb. The inserted gene fragment was compared with the known nucleotide sequence of P116, and the rest was completely identical except for the mutation base.
3, construction and identification of Mp expression vector
The recombinant plasmid was used as a template to amplify a single band with the same length as expected. The recombinant plasmid was cut into two fragments of 0.6kb and 5.8kb with BamHI and EcoRI double enzymes. Two fragments of 0.9kb and 5.5kb were produced by EcoRI and Xhol double enzymes. 1.5kb and two fragments were produced by BamHI, Xhol double enzyme.
4, detection of recombinant protein antigenicity
After induction, the relative molecular mass (Mr) was expressed as a fusion protein of 81KDa, which was consistent with the expected Mr size.Western blotting results. The results showed that the rabbit polyvalent antiserum and purified fusion protein formed a specific antigen antibody reaction band at 81KDa.
conclusion
1, PCR point mutation (TGA to TGG) was used to obtain the fragment of carboxyl terminus of Mp 116 protein.
2, we constructed a P1-P116 double protein multi epitope expression vector and transferred it into E. coli to express the fusion protein stably.
3, Western blotting showed that the fusion protein expressed by 81KDa was immunoreactive.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R375.2

【参考文献】