肺炎嗜衣原體CPAF重組蛋白的表達、純化及其臨床診斷初步應用
發(fā)布時間：2019-07-01 19:34
【摘要】： 目的:構建肺炎嗜衣原體(Chlamydophila pneumoniae, Cpn)蛋白酶樣活性因子(Chlamydial protease like activity factor,CPAF)免疫優(yōu)勢區(qū)(181～400aa, CPAFm)基因的重組表達體,在大腸桿菌中誘導表達,純化表達產(chǎn)物,對其進行免疫活性分析;建立間接ELISA方法檢測Cpn感染臨床標本,評價重組蛋白在臨床診斷中的應用價值,為制備Cpn感染快速診斷試劑盒提供實驗依據(jù)。 方法:通過生物信息學分析并查閱文獻,篩選CPAF免疫優(yōu)勢區(qū)基因,以Cpn CWL029株基因組DNA為模板,高保真PCR擴增目的基因,將其亞克隆至pGEX6p-2原核表達載體中構建重組質粒,經(jīng)酶切和測序鑒定后,轉化至E.coli BL21中,IPTG誘導表達,采用SDS-PAGE和Western blot分析和鑒定表達產(chǎn)物;復性后用谷胱甘肽S-轉移酶(glutathione S-transferase,GST)瓊脂糖凝膠FF純化重組蛋白,A280紫外分光光度法測定純化蛋白濃度。用純化的重組蛋白為包被抗原建立間接ELISA方法,優(yōu)化后進行可靠性試驗和穩(wěn)定性試驗,并檢測其與Ct的交叉反應。以純化的重組蛋白免疫新西蘭兔,用ELISA方法檢測兔血清中特異性抗體效價,分析重組蛋白的免疫原性;用間接ELISA方法和Western blot分析重組蛋白的免疫反應性。間接ELISA方法與金標準方法MIF檢測臨床標本,評價重組蛋白在血清學診斷中的應用價值。以純化的抗重組蛋白多克隆抗體為一抗建立間接ELISA方法,與進口PCR試劑平行檢測呼吸道感染患者痰咽拭子,評價重組蛋白在抗原檢測中的應用價值。 結果:軟件分析CPAF抗原表位并查閱文獻,選擇了CPAF基因第1168241~1167582bp位堿基序列為目的基因(片段長度為660bp,編碼200個氨基酸),PCR擴增得到大小約700bp目的片斷;構建的pGEX6p-2/CPAFm重組質粒經(jīng)酶切和測序鑒定證明插入子為目的基因,測序結果與Genbank上登錄序列完全一致。SDS-PAGE分析顯示,在IPTG誘導下,重組工程菌表達了一相對分子質量(Mr)約51.3×103的目的蛋白,在菌體細胞內(nèi)主要以包涵體形式存在,經(jīng)GST瓊脂糖凝膠FF純化得到純度達95%以上的重組蛋白,Western blot檢測其能與Cpn陽性血清發(fā)生特異性反應。以重組蛋白為包被抗原建立間接ELISA方法,可靠性試驗測得平均批間變異系數(shù)(CV)為6.52%,平均批內(nèi)CV為8.08%;穩(wěn)定性試驗測得平均批間CV為6.93%;檢測Ct陽性參考血清和臨床陽性血清,無一例交叉反應。檢測免疫兔血清中特異性IgG和IgM抗體效價,分別高達1㑳16 000和1㑳8 000以上;檢測Cpn IgG和IgM參考血清,敏感度和特異度均為100%;與MIF試劑同時檢測300份臨床血清標本,IgG和IgM的一致率分別為99.0%和98.3%。以純化的抗重組蛋白多克隆抗體為一抗建立間接ELISA方法,與PCR試劑同時檢測120份病人痰咽拭子中Cpn抗原,一致率為88.3%。 結論: (1)成功構建了pGEX6p-2/CPAFm原核表達重組體,將其轉化至E.coli后表達出一相對分子質量約51.3×103的GST-CPAFm重組蛋白; (2)重組蛋白具有良好的免疫原性,能刺激新西蘭兔產(chǎn)生高效價的特異性IgG和IgM抗體; (3)重組蛋白具有良好的免疫反應性,能與Cpn陽性血清特異性結合,可應用于Cpn感染血清學診斷; (4)重組蛋白在Cpn感染血清學診斷和抗原檢測中具有良好的應用價值。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R374
本文編號：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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R374
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相關期刊論文 前3條
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