抗结核杆菌晶体蛋白人源IgA抗体研制

发布时间：2018-05-24 08:24

本文选题：晶体蛋白 + 结核分枝杆菌　；参考：《广西医科大学》2012年硕士论文

【摘要】：结核分枝杆菌(Mycobacterium tuberculosis, MTB)目前世界上最具致病性的人类病原体。全球目前有30%的人口被MTB感染,其中有90%的感染者以临床上无症状的形式存在,即以潜伏状态存在。结核杆菌晶体蛋白(Alpha-Crystallin, Acr)是结核分枝杆菌潜伏感染期机体免疫反应的重要靶抗原,对MTB进入休眠状态及MTB休眠菌在体内的存活起着重要的作用。本研究选择MTB休眠期重要的保护性抗原a-crystallin(Acr),从大容量人源噬菌体抗体库中筛选特异的抗MTB Acr蛋白的人源抗体。构建IgA真核表达载体,在中国仓鼠卵巢细胞中表达抗结核杆菌的人源IgA抗体,为进一步研究这些抗体对于结核杆菌感染的预防和治疗作用,探索抗晶体蛋白防治结核杆菌感染的可行性和可能的作用机理鉴定基础。 1结核分枝杆菌Acr蛋白表达载体的构建及Acr的表达纯化以MTB H37Rv基因组DNA为模板,通过PCR方法对Acr蛋白的基因进行扩增,以pCold为载体构建重组质粒,再转化到表达宿主菌BL21(DE3)中,以IPTG诱导表达。经SDS-PAGE和Western blotting分析并纯化该表达产物。用超滤的方式将纯化后蛋白质溶液的buffer替换为PBS缓冲液。构建了具有正确基因序列的Acr蛋白重组表达质粒,重组蛋白在大肠杆菌BL21(DE3)中表达。分别用6×his的单克隆抗体(mAb)和抗Acr蛋白mAb对表达产物进行Western blotting分析,结果显示在相对分子量约19kDa处均有特异性条带,与预计大小相吻合,表明成功表达目的蛋白。低温诱导培养,Acr蛋白获得了可溶性表达。纯化、超滤后蛋白纯度达90%,浓度达0.8mg/ml 2结核分枝杆菌Acr蛋白人源抗体的筛选以MTB Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程,从大容量抗体库中筛选特异性(?)(?)MTB Acr蛋白的抗体,并对可变区序列进行了测序分析。将特异性的噬菌体抗体感染HB2151菌,经I PTG诱导表达,制备了抗MTB Acr蛋白的可溶性单链抗体；对其序列和抗原结合活性进行分析鉴定。经过4轮筛选,获得了43个与MTB Acr蛋白结合的阳性克隆,29个特异结合的克隆；测序分析有26不同的可变区片段；通过可溶性scFv表达筛选到14株特异性结合Acr蛋白的可溶性scFv抗体克隆；经过基因测序,分析了可变区基因的亚群。成功制备了可溶性抗体。Westren blotting分析证实筛选的人源抗体能与天然蛋白结合。 3结核分枝杆菌Acr蛋白人源IgA抗体真核表达载体的构建通过融合PCR将重链可变区基因与本室构建的IgA2恒定区的序列拼接,轻链可变区基因与本室构建的k链恒定区基因连接,完成了IgA抗体基因的构建。将IgA抗体克隆到真核表达载体pEF-dhfr-Neo中,构建了pEF-dhfr-IGH,pEF-dhfr-IGK IgA真核表达载体。 4结核分枝杆菌Acr人源IgA抗体在真核细胞中的表达用构建的pEF-dhfr-IGH和pEF-dhfr-IGK表达质粒共转染CHO/dhfr-细胞。72小时后,用鼠抗人的工gA(a)和鼠抗人的kappa链mAb和结核分枝杆菌的Acr蛋白包被酶联板,ELISA检测细胞上清IgA抗体的表达。综上所述：本实验表达出MTB Acr蛋白,通过对大容量噬菌体抗体库的筛选,筛选到14株特异性结合MTB Acr蛋白的可溶性抗体。通过western blotting实验验证了可溶性抗体与天然蛋白的结合活性。构建了抗MTB Acr蛋白的IgA真核表达载体,通过转染中国仓鼠卵巢细胞,获得了工gA抗体,免疫检测结果显示该重组全分子人源IgA抗体具有特异性识别结核杆菌Acr蛋白的能力。
[Abstract]:Mycobacterium tuberculosis (MTB) is currently the most pathogenic human pathogen in the world. 30% of the world's population is currently infected by MTB, of which 90% of the infected people exist in the form of asymptomatic in clinical, that is, the latent state. Mycobacterium tuberculosis (Alpha-Crystallin, Acr) is the potential of Mycobacterium tuberculosis. The important target antigen of the immune response in the stage of volt infection plays an important role in the entry of MTB into dormancy and the survival of MTB dormant bacteria in the body. This study selected the important protective antigen A-crystallin (Acr) of the MTB dormancy period, and screened the human antibody against the specific anti MTB Acr protein from the large human phage antibody library. The expression vector expressed IgA antibody against Mycobacterium tuberculosis in Chinese hamster ovary cells, in order to further study the preventive and therapeutic effects of these antibodies on Mycobacterium tuberculosis infection, and to explore the feasibility and possible mechanism of anti tuberculous bacillus to prevent Mycobacterium tuberculosis infection.
Construction of Mycobacterium tuberculosis Acr protein expression vector 1 and expression and purification of Acr
The MTB H37Rv genome DNA was used as a template to amplify the gene of Acr protein by PCR method, construct the recombinant plasmid with pCold as the carrier, and then convert it into the expression host strain BL21 (DE3) and induce expression in IPTG. The expression product was analyzed and purified by SDS-PAGE and Western blotting. The purified protein solution was replaced by ultrafiltration. The recombinant expression plasmid of Acr protein with correct gene sequence was constructed, and the recombinant protein was expressed in Escherichia coli BL21 (DE3). The expression products were analyzed with 6 x his monoclonal antibody (mAb) and anti Acr protein mAb, respectively. The results showed that there were specific bands at the relative molecular weight around 19kDa. The results showed that the target protein was expressed successfully. The soluble expression of Acr protein was obtained by low temperature induction culture. The purity of protein was 90% after ultrafiltration, and the concentration was up to 0.8mg/ml
Screening of human antibody for 2 Mycobacterium tuberculosis Acr protein
MTB Acr protein was coated with the immune tube, and the antibodies of specific (?) MTB Acr protein were screened from the large capacity antibody library by the 4 rounds of "adsorption elution amplification" of the phage antibody library, and the sequence of the variable region was sequenced. The specific phagocytic antibody was infected with HB2151 bacteria, induced by I PTG, and the anti MTB was prepared. Acr protein soluble single chain antibody; analysis and identification of its sequence and antigen binding activity. After 4 rounds of screening, 43 positive clones combined with MTB Acr protein were obtained, 29 specific clones were cloned, and 26 different variable region fragments were sequenced, and 14 specific binding Acr proteins were screened by soluble scFv expression. The soluble scFv antibody clones were cloned, and the subpopulations of the variable region genes were analyzed by gene sequencing. The soluble antibody.Westren blotting analysis was successfully prepared to confirm that the screened human antibody could be combined with the natural protein.
Construction of eukaryotic expression vector for human IgA antibody of 3 Mycobacterium tuberculosis Acr protein
By splicing the gene of the heavy chain variable region with the sequence of the IgA2 constant region constructed in this room by fusion of PCR, the light chain variable region gene was connected with the K chain constant region gene of the room, and the construction of the IgA antibody gene was completed. The IgA antibody was cloned into the eukaryotic expression vector pEF-dhfr-Neo, and the pEF-dhfr-IGH and pEF-dhfr-IGK IgA eukaryotic expression vector was constructed.
Expression of human IgA antibody against Mycobacterium tuberculosis Acr 4 in eukaryotic cells
After transfecting CHO/dhfr- cells with the constructed pEF-dhfr-IGH and pEF-dhfr-IGK plasmids for.72 hours, the mouse anti human gA (a) and the mouse anti human kappa chain mAb and the Acr protein of Mycobacterium tuberculosis were used to detect the expression of the IgA antibody in the cell supernatant.
To sum up: the MTB Acr protein was expressed in this experiment. By screening the large capacity phage antibody library, 14 soluble antibodies with specific binding of MTB Acr protein were screened. The binding activity of soluble antibody and natural protein was verified by Western blotting experiment. The IgA eukaryotic expression vector of anti MTB Acr protein was constructed and transfected through transfection. The Chinese hamster ovary cells obtained the gA antibody. The results of immunoassay showed that the recombinant human IgA antibody had the ability to identify the Acr protein of Mycobacterium tuberculosis.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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