結(jié)核分枝桿菌保護性抗原的表達、純化及單克隆抗體的制備
發(fā)布時間：2019-04-11 15:22
【摘要】： 結(jié)核病(Tuberculosis, TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB)所致的、以呼吸系統(tǒng)感染為主的慢性傳染病。全球目前約有20億人感染了MTB,每年死于TB的人數(shù)高達200萬。TB已成為人類的頭號傳染病殺手。我國目前至少有5億人感染了MTB,每年死于TB的人數(shù)約有25萬,是我國其他傳染病和寄生蟲病死亡人數(shù)總和的兩倍多�？ń槊�(Bacille Calmette-Guerin,BCG)接種目前仍然是預防TB的主要手段,但總體保護率不穩(wěn)定,介于0%-80%。TB目前仍沒有十分有效的早期診斷方法。隨著MTB耐藥株的出現(xiàn)與人類免疫缺陷病毒(Human immunodeficiency virus HIV)合并感染及人口流動增加等因素的出現(xiàn),進一步加劇了TB對人類的威脅。因此,積極研究MTB致病機制及免疫機制,研制更加有效的診斷及治療MTB感染的新方法、新技術(shù)、新疫苗就具有重大意義。 分泌蛋白是MTB在對數(shù)生長早期分泌到菌體外的一類蛋白,也是目前確認的MTB蛋白中能夠誘發(fā)保護性免疫的一類蛋白,Ag85B和ESAT6是其中重要的組成成分,廣泛用于治療TB的預防和診斷研究中。復活促進因子(Resuscitation promoting factor,RPF)最早是在藤黃微球菌(Micrococcus luteus)中發(fā)現(xiàn)的,它可以促進休眠期MTB的生長。RPFD以分泌形式表達,抗原性較強,因此,RPFD在TB的疫苗研制和快速診斷試劑方面有較大的應用潛力。 實驗目的: 構(gòu)建表達MTB H37RV保護性抗原Ag85B、Ag85B-ESAT6和ESAT6-RPFD的原核表達載體,表達并純化三種蛋白,并制備抗Ag85B蛋白的單克隆抗體(mAb)。 實驗方法和結(jié)果: 1.以MTB H37RV株基因組為模版,采用PCR方法分別擴增ag85B, esat6,rpfD基因,經(jīng)測序證實與Genebank公布的序列完全一致。將目的基因片段亞克隆入pProEXHTb原核表達載體,酶切鑒定陽性重組質(zhì)粒pPro-ag85B、pPro-ag85B-esat6和pPro-esat6-rpfD。用IPTG誘導表達Ag85B、Ag85B-ESAT6和ESAT6-RPFD三種蛋白。SDS-PAGE分析和Western Blot表明,成功地表達了Ag85B、Ag85B-ESAT6和ESAT6-RPFD蛋白。相對分子量分別為32 kD,43 kD和30 kD,與預計蛋白大小相符。用Ni-NTA親和色譜柱變性條件下純化獲得三種重組蛋白,200 mL培養(yǎng)物可分別獲得25.52 mg、17.16 mg和23.76 mg的純化蛋白,得率分別為4.4%、3%和4.1%。 2.取純化的Ag85B蛋白與弗氏不完全佐劑等體積混合,乳化。6周齡雌性BAL/c小鼠100μg/只背部皮下多點注射。間隔2周,共免疫三次。末次免疫兩周后,選擇血清效價高的小鼠再經(jīng)腹腔加強免疫50μg/只。3天后取脾細胞與小鼠骨髓瘤細胞(Sp2/0)融合,加入鋪有飼養(yǎng)細胞的96孔板。用HAT培養(yǎng)液培養(yǎng)1周左右,換成HT培養(yǎng)液用以篩選陽性融合細胞。獲得雜交瘤細胞系1C11,2D5,3H3。經(jīng)測定,這三株雜交瘤細胞系均為IgG1亞類。取效價最高的1C11雜交瘤細胞系注射BAL/c小鼠腹腔,抽取腹水,用硫酸銨法純化腹水。采用Western Blot、間接免疫熒光法和ELISA法測定mAb為抗Ag85B的單克隆抗體,特異性檢測顯示mAb可與MTB發(fā)生特異性反應。
[Abstract]:Tuberculosis (Tuberculosis, TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), mainly respiratory infection. About 2 billion people around the world are infected with MTB, and up to 20 million people die from TB every year. TB has become the number one killer of infectious diseases in humans. At present, there are at least 500 million people infected with MTB, in China, about 250000 of them die from TB every year, which is more than double the total number of deaths of other infectious and parasitic diseases in China. BCG vaccination (Bacille Calmette-Guerin,BCG) is still the main method to prevent TB, but the overall protection rate is not stable. There is still no effective early diagnosis method for 0%-80%.TB. With the emergence of MTB-resistant strains and the co-infection of human immunodeficiency virus (Human immunodeficiency virus HIV) and the increase of population mobility, the threat of TB to human beings is further aggravated. Therefore, it is of great significance to study the pathogenesis and immune mechanism of MTB, to develop a more effective method for diagnosis and treatment of MTB infection, and to develop a new vaccine. Secretory protein is a class of proteins secreted by MTB in the early logarithmic growth phase, and it is also a class of proteins that can induce protective immunity in MTB proteins. Ag85B and ESAT6 are important components of these proteins. It is widely used in the prevention and diagnosis of TB. The resurrection promoter (Resuscitation promoting factor,RPF was first found in the (Micrococcus luteus) of Micrococcus vinifera. It can promote the growth of MTB during dormancy. RPFD is expressed in secretory form and has a strong antigenicity, therefore, the antigenicity of RPFD is stronger. RPFD has great potential in vaccine development and rapid diagnosis of TB. Objective: to construct prokaryotic expression vector expressing MTB H37RV protective antigen Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, express and purify three proteins, and prepare monoclonal antibody (mAb). Against Ag85B protein. Experimental methods and results: 1. Using the genome of MTB H37RV strain as template, the ag85B, esat6,rpfD gene was amplified by PCR and confirmed to be identical with the published sequence of Genebank by sequencing. The target gene fragment was subcloned into the prokaryotic expression vector pProEXHTb, and the positive recombinant plasmids pPro-ag85B,pPro-ag85B-esat6 and pPro-esat6-rpfD. were identified by enzyme digestion. Three proteins, Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, were induced by IPTG. SDS-PAGE analysis and Western Blot showed that Ag85B,Ag85B-ESAT6 and ESAT6-RPFD proteins were successfully expressed. The relative molecular weight was 32 kD,43 kD and 30 kD, respectively, which were consistent with the predicted protein size. Three recombinant proteins were purified by Ni-NTA affinity chromatography column denaturation. The purified proteins of 25.52 mg,17.16 mg and 23.76 mg were obtained by 200 mL culture, and the yields were 4.4%, 3% and 4.1%, respectively. 2. The purified Ag85B protein was mixed with Freund's incomplete adjuvant and emulsified. 6-week-old female BAL/c mice were injected subcutaneously with 100 渭 g / mouse. The mice were immunized three times at a interval of 2 weeks. Two weeks after the last immunization, mice with high serum titer were selected and then immunized with 50 渭 g / mouse intraperitoneally. After 3 days, spleen cells were fused with mouse myeloma cells (Sp2/0), and 96-well plates with feeder cells were added. The cells were cultured in HAT medium for one week or so, then replaced with HT medium for screening positive fusion cells. Hybridoma cell lines 1C11, 2D5, 3H3were obtained. The three hybridoma cell lines were identified as IgG1 subclass. The highest titer 1C11 hybridoma cell line was injected into the abdominal cavity of BAL/c mice, ascites was extracted and purified by ammonium sulfate method. Western Blot, indirect immunofluorescence assay and ELISA method were used to detect mAb as a monoclonal antibody against Ag85B. Specific detection showed that mAb could react specifically with MTB.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2456520
[Abstract]:Tuberculosis (Tuberculosis, TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), mainly respiratory infection. About 2 billion people around the world are infected with MTB, and up to 20 million people die from TB every year. TB has become the number one killer of infectious diseases in humans. At present, there are at least 500 million people infected with MTB, in China, about 250000 of them die from TB every year, which is more than double the total number of deaths of other infectious and parasitic diseases in China. BCG vaccination (Bacille Calmette-Guerin,BCG) is still the main method to prevent TB, but the overall protection rate is not stable. There is still no effective early diagnosis method for 0%-80%.TB. With the emergence of MTB-resistant strains and the co-infection of human immunodeficiency virus (Human immunodeficiency virus HIV) and the increase of population mobility, the threat of TB to human beings is further aggravated. Therefore, it is of great significance to study the pathogenesis and immune mechanism of MTB, to develop a more effective method for diagnosis and treatment of MTB infection, and to develop a new vaccine. Secretory protein is a class of proteins secreted by MTB in the early logarithmic growth phase, and it is also a class of proteins that can induce protective immunity in MTB proteins. Ag85B and ESAT6 are important components of these proteins. It is widely used in the prevention and diagnosis of TB. The resurrection promoter (Resuscitation promoting factor,RPF was first found in the (Micrococcus luteus) of Micrococcus vinifera. It can promote the growth of MTB during dormancy. RPFD is expressed in secretory form and has a strong antigenicity, therefore, the antigenicity of RPFD is stronger. RPFD has great potential in vaccine development and rapid diagnosis of TB. Objective: to construct prokaryotic expression vector expressing MTB H37RV protective antigen Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, express and purify three proteins, and prepare monoclonal antibody (mAb). Against Ag85B protein. Experimental methods and results: 1. Using the genome of MTB H37RV strain as template, the ag85B, esat6,rpfD gene was amplified by PCR and confirmed to be identical with the published sequence of Genebank by sequencing. The target gene fragment was subcloned into the prokaryotic expression vector pProEXHTb, and the positive recombinant plasmids pPro-ag85B,pPro-ag85B-esat6 and pPro-esat6-rpfD. were identified by enzyme digestion. Three proteins, Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, were induced by IPTG. SDS-PAGE analysis and Western Blot showed that Ag85B,Ag85B-ESAT6 and ESAT6-RPFD proteins were successfully expressed. The relative molecular weight was 32 kD,43 kD and 30 kD, respectively, which were consistent with the predicted protein size. Three recombinant proteins were purified by Ni-NTA affinity chromatography column denaturation. The purified proteins of 25.52 mg,17.16 mg and 23.76 mg were obtained by 200 mL culture, and the yields were 4.4%, 3% and 4.1%, respectively. 2. The purified Ag85B protein was mixed with Freund's incomplete adjuvant and emulsified. 6-week-old female BAL/c mice were injected subcutaneously with 100 渭 g / mouse. The mice were immunized three times at a interval of 2 weeks. Two weeks after the last immunization, mice with high serum titer were selected and then immunized with 50 渭 g / mouse intraperitoneally. After 3 days, spleen cells were fused with mouse myeloma cells (Sp2/0), and 96-well plates with feeder cells were added. The cells were cultured in HAT medium for one week or so, then replaced with HT medium for screening positive fusion cells. Hybridoma cell lines 1C11, 2D5, 3H3were obtained. The three hybridoma cell lines were identified as IgG1 subclass. The highest titer 1C11 hybridoma cell line was injected into the abdominal cavity of BAL/c mice, ascites was extracted and purified by ammonium sulfate method. Western Blot, indirect immunofluorescence assay and ELISA method were used to detect mAb as a monoclonal antibody against Ag85B. Specific detection showed that mAb could react specifically with MTB.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
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