肺炎嗜衣原体CPAF重组蛋白的表达、纯化及其临床诊断初步应用
发布时间：2019-07-01 19:34
【摘要】： 目的:构建肺炎嗜衣原体(Chlamydophila pneumoniae, Cpn)蛋白酶样活性因子(Chlamydial protease like activity factor,CPAF)免疫优势区(181～400aa, CPAFm)基因的重组表达体,在大肠杆菌中诱导表达,纯化表达产物,对其进行免疫活性分析;建立间接ELISA方法检测Cpn感染临床标本,评价重组蛋白在临床诊断中的应用价值,为制备Cpn感染快速诊断试剂盒提供实验依据。 方法:通过生物信息学分析并查阅文献,筛选CPAF免疫优势区基因,以Cpn CWL029株基因组DNA为模板,高保真PCR扩增目的基因,将其亚克隆至pGEX6p-2原核表达载体中构建重组质粒,经酶切和测序鉴定后,转化至E.coli BL21中,IPTG诱导表达,采用SDS-PAGE和Western blot分析和鉴定表达产物;复性后用谷胱甘肽S-转移酶(glutathione S-transferase,GST)琼脂糖凝胶FF纯化重组蛋白,A280紫外分光光度法测定纯化蛋白浓度。用纯化的重组蛋白为包被抗原建立间接ELISA方法,优化后进行可靠性试验和稳定性试验,并检测其与Ct的交叉反应。以纯化的重组蛋白免疫新西兰兔,用ELISA方法检测兔血清中特异性抗体效价,分析重组蛋白的免疫原性;用间接ELISA方法和Western blot分析重组蛋白的免疫反应性。间接ELISA方法与金标准方法MIF检测临床标本,评价重组蛋白在血清学诊断中的应用价值。以纯化的抗重组蛋白多克隆抗体为一抗建立间接ELISA方法,与进口PCR试剂平行检测呼吸道感染患者痰咽拭子,评价重组蛋白在抗原检测中的应用价值。 结果:软件分析CPAF抗原表位并查阅文献,选择了CPAF基因第1168241~1167582bp位碱基序列为目的基因(片段长度为660bp,编码200个氨基酸),PCR扩增得到大小约700bp目的片断;构建的pGEX6p-2/CPAFm重组质粒经酶切和测序鉴定证明插入子为目的基因,测序结果与Genbank上登录序列完全一致。SDS-PAGE分析显示,在IPTG诱导下,重组工程菌表达了一相对分子质量(Mr)约51.3×103的目的蛋白,在菌体细胞内主要以包涵体形式存在,经GST琼脂糖凝胶FF纯化得到纯度达95%以上的重组蛋白,Western blot检测其能与Cpn阳性血清发生特异性反应。以重组蛋白为包被抗原建立间接ELISA方法,可靠性试验测得平均批间变异系数(CV)为6.52%,平均批内CV为8.08%;稳定性试验测得平均批间CV为6.93%;检测Ct阳性参考血清和临床阳性血清,无一例交叉反应。检测免疫兔血清中特异性IgG和IgM抗体效价,分别高达1㑳16 000和1㑳8 000以上;检测Cpn IgG和IgM参考血清,敏感度和特异度均为100%;与MIF试剂同时检测300份临床血清标本,IgG和IgM的一致率分别为99.0%和98.3%。以纯化的抗重组蛋白多克隆抗体为一抗建立间接ELISA方法,与PCR试剂同时检测120份病人痰咽拭子中Cpn抗原,一致率为88.3%。 结论: (1)成功构建了pGEX6p-2/CPAFm原核表达重组体,将其转化至E.coli后表达出一相对分子质量约51.3×103的GST-CPAFm重组蛋白; (2)重组蛋白具有良好的免疫原性,能刺激新西兰兔产生高效价的特异性IgG和IgM抗体; (3)重组蛋白具有良好的免疫反应性,能与Cpn阳性血清特异性结合,可应用于Cpn感染血清学诊断; (4)重组蛋白在Cpn感染血清学诊断和抗原检测中具有良好的应用价值。
[Abstract]:Objective: To construct the recombinant expression of the immunodominant region (181-400aa, CPAFm) in the immunodominant region (181-400aa, CPAFm) of the C. pneumoniae (Cpn) protease-like activity factor (CPAF). An indirect ELISA was established to detect the clinical specimens of Cpn infection and to evaluate the application value of recombinant protein in clinical diagnosis and to provide experimental basis for preparing the kit for rapid diagnosis of Cpn infection. Methods: The gene of CPAF immunodominant region was screened by bioinformatics and the gene of CPAF immunodominant region was screened. The target gene was amplified by high-fidelity PCR using the genomic DNA of Cpn CWL029 strain, and then it was subcloned into pGEX6p-2 prokaryotic expression vector to construct the recombinant plasmid. The recombinant protein was purified by SDS-PAGE and Western blot, and the recombinant protein was purified by using glutathione S-transferase (GST). Purified protein concentration. The purified recombinant protein is used as the envelope antigen to establish an indirect ELISA method, the reliability test and the stability test are carried out after the optimization, and the method and the method T cross reaction. The purified recombinant protein was used to immunize New Zealand rabbits. The specific antibody titer in rabbit serum was detected by ELISA. The immunogenicity of recombinant protein was analyzed. The recombinant protein was analyzed by indirect ELISA and Western blot. The indirect ELISA method and the gold standard method MIF for the detection of clinical specimens and the evaluation of the recombinant protein in the serologic diagnosis The application value of the purified anti-recombinant protein polyclonal antibody is an anti-establishment indirect ELISA method, the sputum-throat swabs of the patients with the respiratory tract infection are detected in parallel with the imported PCR reagent, and the recombinant protein is evaluated for detecting the antigen Results: The software was used to analyze the epitope of the CPAF antigen and to review the literature. The target gene (the length of the fragment was 660 bp, the length of the fragment was 660 bp, and the length of the fragment was 200 amino acids) was selected and the PCR was amplified by PCR. The recombinant plasmid pGEX6p-2/ CPAFm was digested and sequenced to prove that the insert was the target gene, and the sequencing result was similar to that of Gen. The results of SDS-PAGE show that, under the induction of IPTG, the recombinant engineering bacteria express a target protein with a relative molecular mass (Mr) of about 51.3-103, which is mainly in the form of inclusion bodies, and is purified by the GST-agarose gel FF. The purity of the recombinant protein is more than 95%, and the Western blot can be used for detecting the recombinant protein with the purity of more than 95% The specific reaction of the positive serum of the pn-positive serum was carried out. The indirect ELISA method was established by using the recombinant protein as the envelope antigen. The mean inter-batch coefficient of variation (CV) was 6.52%, the average intra-batch CV was 8.08%, and the average inter-batch CV was 6.93% in the stability test, and the positive reference serum and the presence of the test were tested. The specific IgG and IgM antibody titres in the serum of the immunized rabbit were as high as 1-16,000 and 1-8,000 respectively. The detection of the serum, the sensitivity and the specific degree of the Cpn IgG and IgM was 100%, and the coincidence rate of the IgG and IgM in 300 clinical serum samples was detected with the MIF reagent. The purified anti-recombinant protein polyclonal antibody is an anti-establishment indirect ELISA method, and 120 patients of sputum and throat swabs of the patients are simultaneously detected by the PCR reagent n-antigen Conclusion: (1) The pGEX6p-2/ CPAFm prokaryotic expression recombinant was successfully constructed and transformed into E. coli to express a relative expression. GST-CPAFm recombinant protein with molecular mass of about 51.3-103; (2) recombinant protein Has good immunogenicity, can stimulate a New Zealand rabbit to generate a high-effective-valent specific IgG and IgM antibody, and (3) a recombinant egg. The white has good immunoreactivity and can be specifically combined with the Cpn positive serum, and can be applied to Cpn.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R374
本文编号：2508742
[Abstract]:Objective: To construct the recombinant expression of the immunodominant region (181-400aa, CPAFm) in the immunodominant region (181-400aa, CPAFm) of the C. pneumoniae (Cpn) protease-like activity factor (CPAF). An indirect ELISA was established to detect the clinical specimens of Cpn infection and to evaluate the application value of recombinant protein in clinical diagnosis and to provide experimental basis for preparing the kit for rapid diagnosis of Cpn infection. Methods: The gene of CPAF immunodominant region was screened by bioinformatics and the gene of CPAF immunodominant region was screened. The target gene was amplified by high-fidelity PCR using the genomic DNA of Cpn CWL029 strain, and then it was subcloned into pGEX6p-2 prokaryotic expression vector to construct the recombinant plasmid. The recombinant protein was purified by SDS-PAGE and Western blot, and the recombinant protein was purified by using glutathione S-transferase (GST). Purified protein concentration. The purified recombinant protein is used as the envelope antigen to establish an indirect ELISA method, the reliability test and the stability test are carried out after the optimization, and the method and the method T cross reaction. The purified recombinant protein was used to immunize New Zealand rabbits. The specific antibody titer in rabbit serum was detected by ELISA. The immunogenicity of recombinant protein was analyzed. The recombinant protein was analyzed by indirect ELISA and Western blot. The indirect ELISA method and the gold standard method MIF for the detection of clinical specimens and the evaluation of the recombinant protein in the serologic diagnosis The application value of the purified anti-recombinant protein polyclonal antibody is an anti-establishment indirect ELISA method, the sputum-throat swabs of the patients with the respiratory tract infection are detected in parallel with the imported PCR reagent, and the recombinant protein is evaluated for detecting the antigen Results: The software was used to analyze the epitope of the CPAF antigen and to review the literature. The target gene (the length of the fragment was 660 bp, the length of the fragment was 660 bp, and the length of the fragment was 200 amino acids) was selected and the PCR was amplified by PCR. The recombinant plasmid pGEX6p-2/ CPAFm was digested and sequenced to prove that the insert was the target gene, and the sequencing result was similar to that of Gen. The results of SDS-PAGE show that, under the induction of IPTG, the recombinant engineering bacteria express a target protein with a relative molecular mass (Mr) of about 51.3-103, which is mainly in the form of inclusion bodies, and is purified by the GST-agarose gel FF. The purity of the recombinant protein is more than 95%, and the Western blot can be used for detecting the recombinant protein with the purity of more than 95% The specific reaction of the positive serum of the pn-positive serum was carried out. The indirect ELISA method was established by using the recombinant protein as the envelope antigen. The mean inter-batch coefficient of variation (CV) was 6.52%, the average intra-batch CV was 8.08%, and the average inter-batch CV was 6.93% in the stability test, and the positive reference serum and the presence of the test were tested. The specific IgG and IgM antibody titres in the serum of the immunized rabbit were as high as 1-16,000 and 1-8,000 respectively. The detection of the serum, the sensitivity and the specific degree of the Cpn IgG and IgM was 100%, and the coincidence rate of the IgG and IgM in 300 clinical serum samples was detected with the MIF reagent. The purified anti-recombinant protein polyclonal antibody is an anti-establishment indirect ELISA method, and 120 patients of sputum and throat swabs of the patients are simultaneously detected by the PCR reagent n-antigen Conclusion: (1) The pGEX6p-2/ CPAFm prokaryotic expression recombinant was successfully constructed and transformed into E. coli to express a relative expression. GST-CPAFm recombinant protein with molecular mass of about 51.3-103; (2) recombinant protein Has good immunogenicity, can stimulate a New Zealand rabbit to generate a high-effective-valent specific IgG and IgM antibody, and (3) a recombinant egg. The white has good immunoreactivity and can be specifically combined with the Cpn positive serum, and can be applied to Cpn.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R374
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相关期刊论文 前3条
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2 倪安平;应当重视衣原体感染的实验室诊断[J];中华检验医学杂志;2005年07期
3 刘钢,王树欣,胡翼云,赵德环,李红丽,江载芳,杨永弘;儿童急性呼吸道感染肺炎衣原体感染状况的研究[J];中国实用儿科杂志;2001年05期
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