本文選題：肺炎支原體 + 蛋白抗原　； 參考：《中國醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 前言 肺炎支原體(Mycoplasma pneumoniae,Mp)是人類原發(fā)性非典型肺炎的病原體,無細胞壁,其引起感染的治療與其它細菌和病毒感染有所不同,因此,Mp感染診斷意義重大。Mp細胞表面的粘附蛋白(如P1,P116等)能與宿主細胞受體結(jié)合,為Mp致病的重要因素,也是引起機體免疫反應(yīng)的主要免疫原,應(yīng)用重組蛋白抗原檢測臨床標(biāo)本,有較高的特異性和敏感性。國內(nèi)外已有人成功構(gòu)建了Mp P1蛋白的表達載體。為了增加重組蛋白的抗原表位,提高診斷的敏感性,本研究經(jīng)PCR點突變技術(shù)(TGA→TGG)獲取Mp116蛋白目的基因,構(gòu)建克隆載體,并與原實驗室構(gòu)建的PGEX-6P-1-P1表達載體重組,構(gòu)建肺炎支原體多抗原表位表達載體,從克隆株中提取純化重組蛋白,經(jīng)Western blotting鑒定融合蛋白的免疫反應(yīng)性,為研制更有效的肺炎支原體基因工程診斷試劑奠定基礎(chǔ)。 方法 1.Mp DNA的制備 按本實驗室常規(guī)方法進行。 2、PCR點突變擴增Mp 116目的基因 根據(jù)GenBank提供的Mp 116蛋白基因(2467—3051)序列,通過引物分析軟件Primer5.0和OLIGO6設(shè)計引物,上游引物序列：5'-TTTTGGATCCTCAAA GATAACATCCAAGTG-3'(BamI),下游引物序列：5'-ATATGAATTCTTGAAGCC CATGTCAGTAAAG-3'(EcoRI),突變引物序列：5'-GACCTTTGGTTGTTTAAGA TTTGGCCTAAGTTC-3'.PCR擴增突變片段,加20μM的突變引物和下游引物以及100ng模板,反應(yīng)總體積50μl,95℃5min×1；95℃30s,55℃30s,72℃40s×30；72℃10min×1�；厥胀蛔兤�,以此做引物擴增目的基因,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%瓊脂糖凝膠電泳鑒定。 3、Mp克隆載體PMD-T-116的構(gòu)建與鑒定 將回收的目的基因和PMD-T載體連接,轉(zhuǎn)入E.coli JM109。通過氨芐青霉素平板篩選陽性克隆株。質(zhì)粒提取試劑盒抽提陽性克隆株的重組質(zhì)粒,PCR擴增及酶切后電泳鑒定。插入的基因序列由南京金思特科技公司測定,并與GenBank提供的FH株肺炎支原體P116核苷酸序列進行同源性比較。 4、Mp多抗原表位表達載體P1-P116的構(gòu)建與鑒定 從克隆株中提取純化重組質(zhì)粒,BamHI、EcoRI雙酶切后進行瓊脂糖凝膠電泳,回收目的片段,與BamHI、EcoRI雙酶切的PGEX-6P-1-P1表達載體(P1表達載體為EcoRI、XhoI雙酶切構(gòu)建)連接,轉(zhuǎn)入E.coli JM109。氨芐青霉素平板篩選轉(zhuǎn)化子,提取純化重組質(zhì)粒,質(zhì)粒酶切圖譜和PCR擴增鑒定。 5、重組蛋白基因表達的檢測與鑒定 重組質(zhì)粒P1-P116轉(zhuǎn)入E.coli BL21中,將陽性克隆株培養(yǎng)菌液接種于含氨芐青霉素(100μg/ml)的LB液體培養(yǎng)基中,37℃振蕩培養(yǎng)過夜。離心收集菌體沉淀,轉(zhuǎn)接至200ml氨芐青霉素LB中,37℃振蕩培養(yǎng)30 min,加入IPTG至終濃度0.5mmol/L,誘導(dǎo)表達2h。離心收集菌體沉淀。冰裕超聲破碎,離心后取上清用Glutathione Sepharose 4B純化GST-P1-P116融合蛋白。SDS-PAGE分析表達產(chǎn)物的相對分子量,免疫印跡實驗鑒定其免疫反應(yīng)性。 結(jié)果 1、P116目的基因片段的擴增與鑒定 PCR擴增的突變引物片段為176bp,擴增的目的基因片段為597bp,經(jīng)1.0%瓊脂糖凝膠電泳鑒定,擴增產(chǎn)物與理論值大小基本相符。 2、PMD-T-116重組質(zhì)粒鑒定 以PMD-T-116為模板,PCR擴增出單一條帶,長度與預(yù)期一致,PMD-T-116用BamHI、EcoRI雙酶切,產(chǎn)生2.7kb和0.6kb兩個片段。插入基因片段與已知的P116核苷酸序列進行比較,除突變堿基外,其余完全一致。 3、Mp表達載體的構(gòu)建與鑒定 以重組質(zhì)粒為模板,PCR擴增出單一條帶,長度與預(yù)期一致。重組質(zhì)粒用BamHI、EcoRI雙酶切產(chǎn)生0.6kb和5.8kb兩個片段,用EcoRI、Xhol雙酶切產(chǎn)生0.9kb和5.5kb兩個片段,用BamHI、Xhol雙酶切產(chǎn)生1.5kb和4.9kb兩個片段。 4、重組蛋白抗原性檢測 經(jīng)誘導(dǎo)后表達相對分子質(zhì)量(Mr)為81KDa的融合蛋白,與預(yù)期的Mr大小一致。Western blotting結(jié)果顯示,兔多價抗血清與純化的融合蛋白在81KDa處形成了一條特異的抗原—抗體反應(yīng)條帶。 結(jié)論 1、本實驗經(jīng)PCR點突變(TGA→TGG)獲取Mp 116蛋白羧基端目的基因片段。 2、構(gòu)建了P1-P116雙蛋白多抗原表位的表達載體,轉(zhuǎn)入大腸桿菌內(nèi),可穩(wěn)定地表達融合蛋白。 3、Western blotting證明表達的81KDa的融合蛋白具有免疫反應(yīng)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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R375.2

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