肺炎衣原體六種包涵體膜蛋白的原核表達(dá)和免疫原性分析
發(fā)布時(shí)間：2018-08-27 10:34
【摘要】： 本課題在對(duì)臨床肺部疾病人群、心腦血管疾病人群以及健康對(duì)照人群進(jìn)行肺炎衣原體感染的初步流行病學(xué)調(diào)查基礎(chǔ)上,進(jìn)一步分析已經(jīng)確定的肺炎衣原體包涵體膜蛋白,即CPn0146、CPn0147、CPn0186、CPn0308、CPn0585和CPn1027在自然人群中的免疫原性,篩選出具有免疫原性的膜蛋白,以期為后期的衣原體的診斷和預(yù)防研究奠定基礎(chǔ)。根據(jù)文獻(xiàn)提供信息選擇六個(gè)編碼肺炎衣原體包涵體膜蛋白的基因,使用Expasy和antheprot2000軟件對(duì)六種基因編碼的蛋白進(jìn)行生物信息學(xué)分析。經(jīng)Expasy分析得出六個(gè)基因編碼蛋白的分子式,摩爾消光系數(shù),半衰期,溶液中的不穩(wěn)定系數(shù),脂肪族指數(shù)、總親水性及修飾位點(diǎn);經(jīng)Antheprot2000分析獲得六個(gè)基因編碼蛋白的抗原性、疏水性、易溶性、親水性、跨膜結(jié)構(gòu)和二級(jí)結(jié)構(gòu)圖。通過比較我們發(fā)現(xiàn)選擇的六種肺炎衣原體包涵體膜蛋白均具有其特征的疏水性跨膜區(qū)域,位于其40-100位氨基酸,由60多個(gè)氨基酸組成。 收集62例心腦血管疾病、88例肺部疾病患者和24例健康對(duì)照人群的血漿標(biāo)本及PBMC標(biāo)本,通過間接ELISA檢測(cè)臨床血漿標(biāo)本中CpnIgG、CpnIgM和CpnIgA三種肺炎衣原體抗體的水平,并通過PCR擴(kuò)增PBMC中編碼16sRNA的CpnDNA,經(jīng)四格表資料的?2檢驗(yàn),比較三組人群中各指標(biāo)的陽性率。在檢測(cè)的62例心腦血管疾病病例中,血漿中CpnIgG、CpnIgM、CpnIgA以及PBMC中Cpn DNA的陽性率分別為67.7%、14.5%、61.3%和71.6%,經(jīng)?2檢驗(yàn),心腦血管疾病人群的CPnIgG、CPnIgA和16SRNA陽性率高于對(duì)照組,P0.05;88例肺部疾患的病例中,血漿中CpnIgG、CpnIgM、CpnIgA以及PBMC中CpnDNA的陽性率分別為50.0%、17.0%、55.6%和82.2%,經(jīng)?2檢驗(yàn),肺部疾病人群的16SRNA明顯高于對(duì)照組,P0.05。說明心腦血管疾病人群的CpnDNA、CPnIgA和CPnIgG陽性率明顯高于健康人群,而在肺部疾病人群僅Cpn DNA陽性率明顯增高,血清學(xué)指標(biāo)無統(tǒng)計(jì)學(xué)意義。同時(shí),通過上述指標(biāo)的檢測(cè)篩選出肺炎衣原體感染的陽性人群和陰性人群,作為下一步肺炎衣原體包涵體膜蛋白的自然人群中免疫原性分析的標(biāo)本。 用PCR法從CPn菌株AR39基因組中擴(kuò)增出六個(gè)編碼包涵體膜蛋白的基因,利用BamHⅠ和NotⅠ進(jìn)行酶切,用T4連接酶與相同酶切處理后的載體pGEX-6P2連接構(gòu)建重組質(zhì)粒,轉(zhuǎn)化入感受體細(xì)菌XL1-blue,通過菌落-PCR、交叉-PCR、序列分析和BLAST對(duì)陽性克隆進(jìn)行鑒定,從而獲得了六種重組質(zhì)粒,序列分析顯示克隆片段的DNA序列與基因庫(kù)中的序列比對(duì)的一致性均為100%;IPTG誘導(dǎo)表達(dá),經(jīng)過GlutathioneSepharose TM 4B純化后,經(jīng)過SDS-PAGE電泳對(duì)表達(dá)的融合蛋白進(jìn)行鑒定,顯示條帶與預(yù)期結(jié)果一致。 選擇表達(dá)較好的蛋白充當(dāng)抗原,使用間接ELISA法測(cè)定Cpn感染者血漿中六種包涵體膜蛋白的抗體水平,進(jìn)一步通過Western-blot確證。間接ELISA結(jié)果顯示,當(dāng)血漿稀釋度為1:500時(shí),抗CPn0147、CPn0308和CPn0186的抗體仍為陽性,Western-blot分析進(jìn)一步證實(shí)了抗體的特異性。說明六種肺炎衣原體包涵體膜蛋白中,CPn0147、CPn0308和CPn0186在自然人群中具有一定的免疫原性。
[Abstract]:Based on the preliminary epidemiological investigation of Chlamydia pneumoniae infection in the population with clinical pulmonary diseases, the population with cardiovascular and cerebrovascular diseases and the healthy control population, the immunogenicity of the identified Chlamydia pneumoniae inclusion body membrane proteins (CPn0146, CPn0147, CPn0186, CPn0308, CPn0585 and CPn1027) in the natural population was further analyzed. Six genes encoding Chlamydia pneumoniae inclusion body membrane proteins were selected according to the literature information. Expasy and antheprot 2000 software were used to analyze the bioinformatics of the proteins encoded by the six genes. The molecular formula, molar extinction coefficient, half-life, instability coefficient, aliphatic index, total hydrophilicity and modification sites of the six gene-encoded proteins were obtained. The antigenicity, hydrophobicity, solubility, hydrophilicity, transmembrane structure and secondary structure diagrams of the six gene-encoded proteins were obtained by Antheprot 2000 analysis. The six inclusion body membrane proteins of Chlamydia pneumoniae all have their own hydrophobic transmembrane region, which is located in the 40-100 amino acids of Chlamydia pneumoniae and consists of more than 60 amino acids.
Plasma samples and PBMC samples from 62 patients with cardiovascular and cerebrovascular diseases, 88 patients with pulmonary diseases and 24 healthy controls were collected. The levels of CpnIgG, CpnIgM and CpnIgA antibodies in clinical plasma samples were detected by indirect ELISA. The CpnDNA encoding 16sRNA in PBMC was amplified by PCR. The results were compared by four-grid table test. The positive rates of CpnIgG, CpnIgM, CpnIgA and Cpn DNA in plasma of 62 patients with cardiovascular and cerebrovascular diseases were 67.7%, 14.5%, 61.3% and 71.6% respectively. The positive rates of CpnIgG, CpnIgM, CpnIgA and PBMC in plasma were 50.0%, 17.0%, 55.6% and 82.2% respectively. The positive rates of 16SRNA in patients with pulmonary diseases were significantly higher than those in control group (P 0.05). The positive rates of CpnDNA, CPnIgA and CPnIgG in patients with cardiovascular and cerebrovascular diseases were significantly higher than those in healthy people, but only in patients with pulmonary diseases. At the same time, the positive and negative groups of Chlamydia pneumoniae infection were screened out through the detection of the above indicators, which can be used as samples for immunogenicity analysis in the next step of Chlamydia pneumoniae inclusion body membrane protein in the natural population.
Six genes encoding inclusion body membrane proteins were amplified from the genome of CPn strain AR39 by PCR. BamH I and Not I were digested and the recombinant plasmid was constructed by T4 ligase and pGEX-6P2. The recombinant plasmid was transformed into XL1-blue. The positive clones were identified by colony-PCR, cross-PCR, sequence analysis and BLAST. Six recombinant plasmids were identified, and the sequence analysis showed that the DNA sequence of the cloned fragment was 100% identical with the sequence alignment in the gene bank; IPTG-induced expression was purified by Glutathione Sepharose TM 4B, and the expressed fusion protein was identified by SDS-PAGE electrophoresis. The results showed that the bands were consistent with the expected results.
The indirect ELISA was used to determine the levels of antibodies against six inclusion body membrane proteins in plasma of Cpn infected patients. The results of indirect ELISA showed that the antibodies against CPn0147, CPn0308 and CPn0186 were still positive when the plasma dilution was 1:500. Western blot analysis further confirmed that the antibodies against CPn0147, CPn0308 and CPn0186 were positive. The specificity of the antibody showed that CPn0147, CPn0308 and CPn0186 were immunogenic to some extent among the six Chlamydia pneumoniae inclusion body membrane proteins.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
本文編號(hào)：2207016
[Abstract]:Based on the preliminary epidemiological investigation of Chlamydia pneumoniae infection in the population with clinical pulmonary diseases, the population with cardiovascular and cerebrovascular diseases and the healthy control population, the immunogenicity of the identified Chlamydia pneumoniae inclusion body membrane proteins (CPn0146, CPn0147, CPn0186, CPn0308, CPn0585 and CPn1027) in the natural population was further analyzed. Six genes encoding Chlamydia pneumoniae inclusion body membrane proteins were selected according to the literature information. Expasy and antheprot 2000 software were used to analyze the bioinformatics of the proteins encoded by the six genes. The molecular formula, molar extinction coefficient, half-life, instability coefficient, aliphatic index, total hydrophilicity and modification sites of the six gene-encoded proteins were obtained. The antigenicity, hydrophobicity, solubility, hydrophilicity, transmembrane structure and secondary structure diagrams of the six gene-encoded proteins were obtained by Antheprot 2000 analysis. The six inclusion body membrane proteins of Chlamydia pneumoniae all have their own hydrophobic transmembrane region, which is located in the 40-100 amino acids of Chlamydia pneumoniae and consists of more than 60 amino acids.
Plasma samples and PBMC samples from 62 patients with cardiovascular and cerebrovascular diseases, 88 patients with pulmonary diseases and 24 healthy controls were collected. The levels of CpnIgG, CpnIgM and CpnIgA antibodies in clinical plasma samples were detected by indirect ELISA. The CpnDNA encoding 16sRNA in PBMC was amplified by PCR. The results were compared by four-grid table test. The positive rates of CpnIgG, CpnIgM, CpnIgA and Cpn DNA in plasma of 62 patients with cardiovascular and cerebrovascular diseases were 67.7%, 14.5%, 61.3% and 71.6% respectively. The positive rates of CpnIgG, CpnIgM, CpnIgA and PBMC in plasma were 50.0%, 17.0%, 55.6% and 82.2% respectively. The positive rates of 16SRNA in patients with pulmonary diseases were significantly higher than those in control group (P 0.05). The positive rates of CpnDNA, CPnIgA and CPnIgG in patients with cardiovascular and cerebrovascular diseases were significantly higher than those in healthy people, but only in patients with pulmonary diseases. At the same time, the positive and negative groups of Chlamydia pneumoniae infection were screened out through the detection of the above indicators, which can be used as samples for immunogenicity analysis in the next step of Chlamydia pneumoniae inclusion body membrane protein in the natural population.
Six genes encoding inclusion body membrane proteins were amplified from the genome of CPn strain AR39 by PCR. BamH I and Not I were digested and the recombinant plasmid was constructed by T4 ligase and pGEX-6P2. The recombinant plasmid was transformed into XL1-blue. The positive clones were identified by colony-PCR, cross-PCR, sequence analysis and BLAST. Six recombinant plasmids were identified, and the sequence analysis showed that the DNA sequence of the cloned fragment was 100% identical with the sequence alignment in the gene bank; IPTG-induced expression was purified by Glutathione Sepharose TM 4B, and the expressed fusion protein was identified by SDS-PAGE electrophoresis. The results showed that the bands were consistent with the expected results.
The indirect ELISA was used to determine the levels of antibodies against six inclusion body membrane proteins in plasma of Cpn infected patients. The results of indirect ELISA showed that the antibodies against CPn0147, CPn0308 and CPn0186 were still positive when the plasma dilution was 1:500. Western blot analysis further confirmed that the antibodies against CPn0147, CPn0308 and CPn0186 were positive. The specificity of the antibody showed that CPn0147, CPn0308 and CPn0186 were immunogenic to some extent among the six Chlamydia pneumoniae inclusion body membrane proteins.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 ;Characterization of Chlamydia trachomatis omp1 gene among sexually transmitted disease patients in south China[J];Journal of Microbiology and Immunology;2005年03期
2 賈天軍;劉殿武;羅建華;鐘光明;;沙眼衣原體CT-249基因編碼蛋白為一包涵體膜蛋白[J];微生物學(xué)報(bào);2007年04期
3 賈天軍;劉殿武;羅建華;張庶民;鐘光明;;肺炎衣原體CPn0308的基因克隆及其內(nèi)源性蛋白定位的研究[J];衛(wèi)生研究;2008年02期
，本文編號(hào)：2207016
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