结核分枝杆菌MPT64-Rv1985c融合蛋白的诊断作用及其重组卡介苗构建的研究

发布时间：2018-07-29 14:35

【摘要】：目的：构建pET32a(+)-MPT64-Rv1985c重组蛋白表达载体,并在大肠杆菌BL21(DE3)菌株中表达,通过血清和胸腔积液样本研究目的蛋白诊断特性；构建PUV15-MPT64-Rv1985c穿梭载体,并电转入卡介苗构建重组卡介苗,为验证融合蛋白保护特性提供准备。方法：通过PCR反应以结核分枝杆菌标准株H37Rv基因组为模板分别获取MPT64和Rv1985c基因片段后,通过重组PCR技术将两基因融合成为MPT64-Rv1985c重组基因,将目的基因经EcoRI和HindⅢ双酶切连入pET32a(+)载体中,经1mM IPTG,37℃过夜诱导,目的蛋白在大肠杆菌BL21(DE3)中表达；应用镍柱纯化目的蛋白,并通过梯度尿素复性。分别以PPD阴性血清和非结核胸腔积液样本的OD450平均值加2倍的标准差作为各自临界值,运用ELISA方法检测211例血清和182例胸腔积液样本中IgG抗体应答水平判断融合蛋白诊断特性。随后将目的基因片段经SphI和NheⅠ双酶切连入PUV15穿梭载体中,并通过电击转化入卡介苗中构建重组卡介苗,经RT-PCR扩增和Western blot对重组卡介苗进行鉴定。结果：成功构建pET32a(+)-MPT64, pET32a(+)-Rv1985c, pET32a(+)-MPT64-Rv1985c重组载体,在大肠杆菌BL21(DE3)中目的蛋白均以包涵体形式表达,经镍柱纯化并通过梯度尿素成功获得大小为42kDa,52kDa,76kDa天然结构目的蛋白；通过ELISA方法检测211例血清中IgG抗体应答水平研究显示,融合蛋白检测活动性结核和PPD阴性对照组的敏感性和特异性分别为50%和93%。较MPT64蛋白和Rv1985c蛋白的敏感性32.9%和35.5%分别增加了17.1%和14.5%。MPT64-Rv1985c蛋白运用ELISA方法检测IgG抗体检测肺结核、肺外结核、活动性结核组及PPD阳性组中的敏感性为41.9%、55.6%、50%和15.4%,特异性为93%。在一定程度上,能有效区分肺结核和肺外结核样本(P=0.014),在肺结核和PPD阳性组以及肺外结核和PPD阳性组别均有显著的差异,证实融合蛋白在区分PPD阳性组别中具有很好的诊断效果；同时验证MPT64蛋白可以在一定程度上区分PPD阳性组别和活动性结核患者,但在区分肺结核和肺外结核患者,PPD阳性组和PPD阴性组时,没有显著的差异；Rv1985c蛋白的血清学检测显示,Rv1985c可显著区分肺结核与PPD阳性及PPD阴性组,是诊断肺结核的一个优选抗原。通过182例胸腔积液样本的IgG抗体应答研究显示,MPT64-Rv1985c蛋白在结核组和非结核组胸腔积液样本检测的敏感性和特异性分别为54.9%和91.2%,比较MPT64蛋白的敏感性和特异性26.3%,95.6%和Rv1985c蛋白检测结核组的敏感性和特异性48.3%和93.4%,敏感性分别增加了28.6%和6.6%,但特异性下降了4.4%和2.2%。统计学分析融合蛋白可以显著区分结核组和非结核组胸腔积液样本(P0.001);受试工作者特征曲线分析显示出MPT64-Rv1985c融合蛋白的诊断效果要优越于单一抗原。成功构建PUV15-MPT64, PUV15-Rv1985c, PUV15-MPT64-Rv1985c穿梭载体,经电击转化入卡介苗,获得PUV15空载体、MPT64和MPT64-Rv1985c重组卡介苗菌株,但未获得Rv1985c重组卡介苗。经RT-PCR和Western blot验证在27kDa,51kDa,85kDa处有蛋白条带产生,分析确定MPT64蛋白和MPT64-Rv1985c融合蛋白能在卡介苗中表达。结论：MPT64-Rv1985c融合蛋白能提高单一抗原体液免疫诊断特性,具有作为新型结核病诊断试剂的潜能。成功获得重组MPT64-Rv1985c-BCG菌株,为验证融合蛋白保护原性提供准备。
[Abstract]:Objective: to construct pET32a (+) -MPT64-Rv1985c recombinant protein expression vector, and to express in Escherichia coli BL21 (DE3) strain, to study the diagnostic characteristics of the target protein through serum and pleural effusion samples, construct PUV15-MPT64-Rv1985c shuttle vector, and turn into BCG vaccine to construct a heavy group of BCG vaccine, in order to provide preparation for the protection of fusion protein.
Methods: the MPT64 and Rv1985c gene fragments were obtained by PCR reaction using the H37Rv genome of Mycobacterium tuberculosis standard strain, and the two gene was fused into MPT64-Rv1985c recombinant gene by recombinant PCR technology. The target gene was joined into pET32a (+) vector by EcoRI and Hind III double enzymes. The target egg was induced by 1mM IPTG, 37 degrees centigrade for the night. The expression of white in Escherichia coli BL21 (DE3) was used to purify the target protein with a nickel column, and through the gradient urea refolding. The standard deviation of the OD450 average of PPD negative sera and non tuberculous pleural effusions was added respectively as their respective critical values. The IgG antibody response levels in 211 cases of serum and 182 cases of pleural effusion were detected by ELISA method. The diagnostic characteristics of the fusion protein were obtained. The target gene fragment was subsequently cut into the PUV15 shuttle vector by SphI and Nhe I, and the recombinant BCG was constructed by electric shock conversion into BCG, and the recombinant BCG was identified by RT-PCR amplification and Western blot.
Results: pET32a (+) -MPT64, pET32a (+) -Rv1985c, pET32a (+) -MPT64-Rv1985c recombinant vector was successfully constructed. The target proteins were expressed in the form of inclusion body in Escherichia coli BL21 (DE3), and purified by the nickel column and successfully obtained by the gradient urea, the size of the protein was 42kDa, 52kDa, and 76kDa natural structure, and 211 serum samples were detected by ELISA method. The sensitivity and specificity of the IgG antibody response level showed that the sensitivity and specificity of the fusion protein were 50% and the sensitivity of the PPD negative control group were 50% and the sensitivity 32.9% and 35.5% of the MPT64 protein and Rv1985c protein were increased by 17.1% and 14.5%.MPT64-Rv1985c, respectively, and the ELISA method was used to detect IgG antibody in the detection of pulmonary tuberculosis, extrapulmonary tuberculosis, and survival. The sensitivity of the active tuberculosis group and the PPD positive group was 41.9%, 55.6%, 50% and 15.4%, and the specificity of 93%. could effectively distinguish between pulmonary tuberculosis and extrapulmonary tuberculosis (P=0.014). There were significant differences in the tuberculosis and PPD positive group, the pulmonary tuberculosis and the PPD positive group, which confirmed the fusion protein in the PPD positive group. MPT64 protein can distinguish between PPD positive and active tuberculosis to some extent, but there is no significant difference between the PPD positive group and the PPD negative group in the diagnosis of tuberculosis and extrapulmonary tuberculosis; the serological detection of Rv1985c protein shows that Rv1985c can significantly distinguish between tuberculosis and PPD positive and PPD positive. The PPD negative group is a preferred antigen for the diagnosis of pulmonary tuberculosis. The sensitivity and specificity of MPT64-Rv1985c protein detection in the tuberculous and non tuberculous group of pleural effusion samples were 54.9% and 91.2%, respectively, by the IgG antibody response study in 182 cases of pleural effusion. The sensitivity and specificity of MPT64 protein were compared with 26.3%, 95.6% and Rv1985c protein. The sensitivity and specificity of the tuberculosis group were 48.3% and 93.4%, the sensitivity increased by 28.6% and 6.6%, respectively, but the specificity decreased by 4.4% and 2.2%.. The fusion protein could distinguish the pleural effusion samples (P0.001) in the tuberculosis group and non tuberculosis group (P0.001). The characteristic curve analysis of the subjects showed the diagnostic effect of MPT64-Rv1985c fusion protein. The fruit should be superior to the single antigen. A successful construction of PUV15-MPT64, PUV15-Rv1985c, PUV15-MPT64-Rv1985c shuttle carrier, converted into BCG by electric shock, PUV15 empty carrier, MPT64 and MPT64-Rv1985c recombinant Bacillus Calmette vaccine, but no Rv1985c recombinant Bacillus Calmette vaccine was obtained. It was confirmed by RT-PCR and Western blot that there was a protein band in 27kDa, 51kDa. MPT64 protein and MPT64-Rv1985c fusion protein can be expressed in BCG.
Conclusion: MPT64-Rv1985c fusion protein can improve the characteristics of the single antigen humoral immune diagnosis, and it has the potential as a new diagnostic reagent for tuberculosis. The recombinant MPT64-Rv1985c-BCG strain can be successfully obtained to provide preparation for the protection of the fusion protein.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【相似文献】