【摘要】：目的：構(gòu)建pET32a(+)-MPT64-Rv1985c重組蛋白表達(dá)載體,并在大腸桿菌BL21(DE3)菌株中表達(dá),通過血清和胸腔積液樣本研究目的蛋白診斷特性；構(gòu)建PUV15-MPT64-Rv1985c穿梭載體,并電轉(zhuǎn)入卡介苗構(gòu)建重組卡介苗,為驗(yàn)證融合蛋白保護(hù)特性提供準(zhǔn)備。 方法：通過PCR反應(yīng)以結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv基因組為模板分別獲取MPT64和Rv1985c基因片段后,通過重組PCR技術(shù)將兩基因融合成為MPT64-Rv1985c重組基因,將目的基因經(jīng)EcoRI和HindⅢ雙酶切連入pET32a(+)載體中,經(jīng)1mM IPTG,37℃過夜誘導(dǎo),目的蛋白在大腸桿菌BL21(DE3)中表達(dá)；應(yīng)用鎳柱純化目的蛋白,并通過梯度尿素復(fù)性。分別以PPD陰性血清和非結(jié)核胸腔積液樣本的OD450平均值加2倍的標(biāo)準(zhǔn)差作為各自臨界值,運(yùn)用ELISA方法檢測(cè)211例血清和182例胸腔積液樣本中IgG抗體應(yīng)答水平判斷融合蛋白診斷特性。隨后將目的基因片段經(jīng)SphI和NheⅠ雙酶切連入PUV15穿梭載體中,并通過電擊轉(zhuǎn)化入卡介苗中構(gòu)建重組卡介苗,經(jīng)RT-PCR擴(kuò)增和Western blot對(duì)重組卡介苗進(jìn)行鑒定。 結(jié)果：成功構(gòu)建pET32a(+)-MPT64, pET32a(+)-Rv1985c, pET32a(+)-MPT64-Rv1985c重組載體,在大腸桿菌BL21(DE3)中目的蛋白均以包涵體形式表達(dá),經(jīng)鎳柱純化并通過梯度尿素成功獲得大小為42kDa,52kDa,76kDa天然結(jié)構(gòu)目的蛋白；通過ELISA方法檢測(cè)211例血清中IgG抗體應(yīng)答水平研究顯示,融合蛋白檢測(cè)活動(dòng)性結(jié)核和PPD陰性對(duì)照組的敏感性和特異性分別為50%和93%。較MPT64蛋白和Rv1985c蛋白的敏感性32.9%和35.5%分別增加了17.1%和14.5%。MPT64-Rv1985c蛋白運(yùn)用ELISA方法檢測(cè)IgG抗體檢測(cè)肺結(jié)核、肺外結(jié)核、活動(dòng)性結(jié)核組及PPD陽性組中的敏感性為41.9%、55.6%、50%和15.4%,特異性為93%。在一定程度上,能有效區(qū)分肺結(jié)核和肺外結(jié)核樣本(P=0.014),在肺結(jié)核和PPD陽性組以及肺外結(jié)核和PPD陽性組別均有顯著的差異,證實(shí)融合蛋白在區(qū)分PPD陽性組別中具有很好的診斷效果；同時(shí)驗(yàn)證MPT64蛋白可以在一定程度上區(qū)分PPD陽性組別和活動(dòng)性結(jié)核患者,但在區(qū)分肺結(jié)核和肺外結(jié)核患者,PPD陽性組和PPD陰性組時(shí),沒有顯著的差異；Rv1985c蛋白的血清學(xué)檢測(cè)顯示,Rv1985c可顯著區(qū)分肺結(jié)核與PPD陽性及PPD陰性組,是診斷肺結(jié)核的一個(gè)優(yōu)選抗原。通過182例胸腔積液樣本的IgG抗體應(yīng)答研究顯示,MPT64-Rv1985c蛋白在結(jié)核組和非結(jié)核組胸腔積液樣本檢測(cè)的敏感性和特異性分別為54.9%和91.2%,比較MPT64蛋白的敏感性和特異性26.3%,95.6%和Rv1985c蛋白檢測(cè)結(jié)核組的敏感性和特異性48.3%和93.4%,敏感性分別增加了28.6%和6.6%,但特異性下降了4.4%和2.2%。統(tǒng)計(jì)學(xué)分析融合蛋白可以顯著區(qū)分結(jié)核組和非結(jié)核組胸腔積液樣本(P0.001);受試工作者特征曲線分析顯示出MPT64-Rv1985c融合蛋白的診斷效果要優(yōu)越于單一抗原。成功構(gòu)建PUV15-MPT64, PUV15-Rv1985c, PUV15-MPT64-Rv1985c穿梭載體,經(jīng)電擊轉(zhuǎn)化入卡介苗,獲得PUV15空載體、MPT64和MPT64-Rv1985c重組卡介苗菌株,但未獲得Rv1985c重組卡介苗。經(jīng)RT-PCR和Western blot驗(yàn)證在27kDa,51kDa,85kDa處有蛋白條帶產(chǎn)生,分析確定MPT64蛋白和MPT64-Rv1985c融合蛋白能在卡介苗中表達(dá)。 結(jié)論：MPT64-Rv1985c融合蛋白能提高單一抗原體液免疫診斷特性,具有作為新型結(jié)核病診斷試劑的潛能。成功獲得重組MPT64-Rv1985c-BCG菌株,為驗(yàn)證融合蛋白保護(hù)原性提供準(zhǔn)備。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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