肺炎衣原体六种包涵体膜蛋白的原核表达和免疫原性分析

发布时间：2018-08-27 10:34

【摘要】： 本课题在对临床肺部疾病人群、心脑血管疾病人群以及健康对照人群进行肺炎衣原体感染的初步流行病学调查基础上,进一步分析已经确定的肺炎衣原体包涵体膜蛋白,即CPn0146、CPn0147、CPn0186、CPn0308、CPn0585和CPn1027在自然人群中的免疫原性,筛选出具有免疫原性的膜蛋白,以期为后期的衣原体的诊断和预防研究奠定基础。根据文献提供信息选择六个编码肺炎衣原体包涵体膜蛋白的基因,使用Expasy和antheprot2000软件对六种基因编码的蛋白进行生物信息学分析。经Expasy分析得出六个基因编码蛋白的分子式,摩尔消光系数,半衰期,溶液中的不稳定系数,脂肪族指数、总亲水性及修饰位点;经Antheprot2000分析获得六个基因编码蛋白的抗原性、疏水性、易溶性、亲水性、跨膜结构和二级结构图。通过比较我们发现选择的六种肺炎衣原体包涵体膜蛋白均具有其特征的疏水性跨膜区域,位于其40-100位氨基酸,由60多个氨基酸组成。收集62例心脑血管疾病、88例肺部疾病患者和24例健康对照人群的血浆标本及PBMC标本,通过间接ELISA检测临床血浆标本中CpnIgG、CpnIgM和CpnIgA三种肺炎衣原体抗体的水平,并通过PCR扩增PBMC中编码16sRNA的CpnDNA,经四格表资料的?2检验,比较三组人群中各指标的阳性率。在检测的62例心脑血管疾病病例中,血浆中CpnIgG、CpnIgM、CpnIgA以及PBMC中Cpn DNA的阳性率分别为67.7%、14.5%、61.3%和71.6%,经?2检验,心脑血管疾病人群的CPnIgG、CPnIgA和16SRNA阳性率高于对照组,P0.05;88例肺部疾患的病例中,血浆中CpnIgG、CpnIgM、CpnIgA以及PBMC中CpnDNA的阳性率分别为50.0%、17.0%、55.6%和82.2%,经?2检验,肺部疾病人群的16SRNA明显高于对照组,P0.05。说明心脑血管疾病人群的CpnDNA、CPnIgA和CPnIgG阳性率明显高于健康人群,而在肺部疾病人群仅Cpn DNA阳性率明显增高,血清学指标无统计学意义。同时,通过上述指标的检测筛选出肺炎衣原体感染的阳性人群和阴性人群,作为下一步肺炎衣原体包涵体膜蛋白的自然人群中免疫原性分析的标本。用PCR法从CPn菌株AR39基因组中扩增出六个编码包涵体膜蛋白的基因,利用BamHⅠ和NotⅠ进行酶切,用T4连接酶与相同酶切处理后的载体pGEX-6P2连接构建重组质粒,转化入感受体细菌XL1-blue,通过菌落-PCR、交叉-PCR、序列分析和BLAST对阳性克隆进行鉴定,从而获得了六种重组质粒,序列分析显示克隆片段的DNA序列与基因库中的序列比对的一致性均为100%;IPTG诱导表达,经过GlutathioneSepharose TM 4B纯化后,经过SDS-PAGE电泳对表达的融合蛋白进行鉴定,显示条带与预期结果一致。选择表达较好的蛋白充当抗原,使用间接ELISA法测定Cpn感染者血浆中六种包涵体膜蛋白的抗体水平,进一步通过Western-blot确证。间接ELISA结果显示,当血浆稀释度为1:500时,抗CPn0147、CPn0308和CPn0186的抗体仍为阳性,Western-blot分析进一步证实了抗体的特异性。说明六种肺炎衣原体包涵体膜蛋白中,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.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

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