牛分枝杆菌主要分泌性蛋白70和脂蛋白83的克隆表达及抗原性研究

发布时间：2018-06-07 21:57

本文选题：牛分枝杆菌 + 主要分泌型免疫蛋白(MPB70)　；参考：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 自1882年德国细菌学家Koch发现结核分枝杆菌以来，已报道的分枝杆菌有100多种。国际上通常将分枝杆菌分为典型结核分枝杆菌(Tuberculosismycobacterium，TM)和非结核分枝杆菌(Non-tuberculosis mycobacterium, NTM)。TM包括结核分枝杆菌、牛分枝杆菌、非洲结核分枝杆菌，又称为结核分枝杆菌复合群( Mycobacterium tuberculosis complex，MTBC)，NTM指MTBC和麻风分枝杆菌以外的其它所有分枝杆菌。牛分枝杆菌可在人与动物之间传播，近年来发现一部分人类结核是由牛分枝杆菌引起并有增多的趋势，已成为值得关注的公共卫生问题。牛分枝杆菌有广泛的宿主，最为常见的是牛、獾和鹿。研究表明，感染牛分枝杆菌的动物可通过痰液、乳汁、粪便等向外排菌而污染环境，因此，控制动物结核是控制人类结核的重要环节。牛结核的检疫方法目前大多使用牛分枝杆菌纯蛋白衍生物（PPD-M）皮肤敏感试验（TST），48小时才能得出试验结果，但PPD中许多抗原成分为非致病性环境分枝杆菌和卡介苗（BCG）所共有，并不能区别非致病性分枝杆菌和牛分枝杆菌感染，检测结果可能出现假阳性。胶体金免疫层析方法有方便，快速等诸多优点，近年来受到广泛关注，胶体金是指氯金酸在一些还原剂如维生素C、柠檬酸钠等的作用下所形成特定大小的金颗粒。在适宜pH条件下，胶体金颗粒表面带负电荷，可与蛋白质所带正电核基团形成非共价键而牢固结合，，这种结合对蛋白的生物学活性无明显影响。胶体金颗粒对蛋白质有较强的吸附作用，可与葡萄球菌A蛋白(SPA)、免疫球蛋白等诸多生物大分子非共价结合。牛分枝杆菌主要分泌性免疫蛋白70(MPB70)和表面脂蛋白83(MPB83)是其两个主要抗原成分，在感染早期诱导机体产生细胞免疫和体液免疫，且抗体在血清中的含量与疾病发展呈正相关。虽然结核分枝杆菌也存在这两个抗原成分，但表达量极低，因此，检测MPB70和MPB83抗体对于牛分枝杆菌感染诊断具有重要意义。本研究采用聚合酶链反应（polymerse chain reaction，PCR）从牛分枝杆菌基因组中扩增mpb70和mpb83基因片段，分别插入原核质粒pET-32a(+)，获得重组表达质粒pET-32a(+)-mpb70和pET-32a(+)-mpb83，转化感受态细胞后，选取阳性克隆，阳性克隆经测序表明目的基因已插入表达质粒，核苷酸序列与牛分枝杆菌标准株AF2122/97同源性为100%。经过表达条件的摸索，在1mM IPTG 37℃诱导3小时，可获得rMPB70和rMPB83高效表达，目的蛋白约占菌体总蛋白的60%以上，且目的蛋白以包涵体表达为主。pET-32a(+)表达载体因融合有硫氧还蛋白和S蛋白，预测rMPB70分子量约为42kDa，rMPB83分子量约为45kDa，SDS-PAGE分析rMPB70和rMPB83大小与预测结果相符。rMPB70和rMPB83蛋白包涵体用尿素变性，半透膜透析去除尿素复性蛋白，rMPB70和rMPB83纯化后蛋白纯度可达80%以上，WB实验分析表明，rMPB70和rMPB83蛋白可与结核病牛血清发生特异反应，说明rMPB70和rMPB83蛋白具有较好的抗原性。本研究探索了rMPB70和rMPB83在胶体金免疫层析试剂中的初步应用，用柠檬酸三钠作为还原剂来制备胶体金颗粒，并利用SPA能与多种哺乳动物IgG结合的特性，用胶体金标记的SPA制备免疫探针，用不同浓度的rMPB70和rMPB83蛋白分别作为靶抗原致敏硝酸纤维素膜，确定rMPB70和rMPB83蛋白的最佳致敏浓度分别为4.5mg/ml、3mg/ml。组装试纸条，并分别用TST阳性牛血清；正常牛血清和生理盐水层析试纸条，15min后观察结果。实验室初步考核显示，用rMPB70蛋白制备的胶体金试剂检测30份TST阳性牛血清，20份呈阳性，敏感性66%；用rMPB83蛋白制备的的胶体金试剂检测30份TST阳性牛血清，22份呈阳性，敏感性73%。而分别检测正常牛血清和生理盐水均呈阴性，显示本研究获得的rMPB70，rMPB83蛋白可以作为捕获抗原用于制备牛结核抗体免疫胶体金试剂。
[Abstract]:The M.tuberculosis has been reported to have more than 100 Mycobacterium tuberculosis since the discovery of Mycobacterium tuberculosis by the German bacteriologist Koch . In the world , Mycobacterium tuberculosis is generally divided into Mycobacterium tuberculosis ( TM ) and Non - tuberculosis Mycobacterium tuberculosis . TM includes Mycobacterium tuberculosis , Mycobacterium tuberculosis , Mycobacterium tuberculosis , also known as Mycobacterium tuberculosis complex ( MTBC ) , which refers to all other Mycobacteria other than MTBC and M . M . M .

In recent years , it has been found that a part of human tuberculosis is caused by Mycobacterium species and has an increased tendency . It has become an important part of the control of human tuberculosis .

The colloidal gold immunochromatography method has many advantages , such as convenient , rapid and so on . In recent years , the colloidal gold refers to gold particles of certain size under the action of some reducing agents such as vitamin C , sodium citrate and the like .

The main secretory immune protein 70 ( MPB70 ) and the surface lipoprotein 83 ( MPB83 ) are two major antigenic components , which induce cellular immunity and humoral immunity at the early stage of infection , and the content of the antibody in serum is positively correlated with the development of disease . Although Mycobacterium tuberculosis also has the two antigenic components , the expression amount is very low , so the detection of MPB70 and MPB83 antibodies has important significance for the diagnosis of Mycobacterium tuberculosis infection .

Recombinant expression plasmid pET - 32a ( + ) - mpb70 and pET - 32a ( + ) - mpb83 were inserted into pET - 32a ( + ) - mpb70 and pET - 32a ( + ) - mpb83 respectively .

In this study , the preliminary application of rMPB70 and rMPB83 in colloidal gold immunochromatography reagent was explored . The colloidal gold particles were prepared by using trisodium citrate as reducing agent , and SPA can be used as the reducing agent to prepare the colloidal gold particles . Using SPA to prepare the immune probe , the optimal sensitizing concentration of rMPB70 and rMPB83 protein was 4.5 mg / ml and 3 mg / ml respectively .
The results were observed after 15 min of normal bovine serum and normal saline chromatography . The results showed that the colloidal gold reagent prepared by rMPB70 protein was positive and sensitive 66 % .
The results showed that the rMPB70 and rMPB83 protein could be used as capture antigen to prepare immune colloidal gold reagent for bovine tuberculosis antibody .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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