结核分枝杆菌lhp基因的原核表达及其产物的单克隆抗体研制

发布时间：2018-12-31 15:03

【摘要】： 结核病是一种人兽共患的慢性消耗性传染病,已成为传染病中的第一号杀手和最大的死亡原因。特别在发展中国家结核病对于感染免疫缺陷病毒的人群来说尤为严重。随着艾滋病患者人数的增加、耐药性菌株的产生,结核病的发病率有逐年增加的趋势。因此,对于病情的流行,早期的感染诊断很关键,急需进一步改进现有的诊断方法。lhp基因是结核分枝杆菌特异性基因,在疫苗株BCG及环境中的非结核分枝杆菌中缺失。lhp与细菌的毒力相关,其缺失可以降低结核分枝杆菌的毒力。CFP-10是lhp的表达蛋白。本研究构建了原核表达CFP-10蛋白的重组大肠杆菌,获得具有生物活性的融合蛋白,并且制备了CFP-10特异性单克隆抗体。这些结果为研究结核分枝杆菌致病机理及免疫机理提供了重要条件,同时,为结核病诊断与检疫提供了新的试剂。一、结核分枝杆菌lhp基因的原核表达 CFP-10作为免疫优势抗原,其功能研究可以加深对结核分枝杆致病机制、免疫机制的研究。本研究采用PCR及GeneSOEing技术分别扩增出lhp基因及lhp-linker-esat-6片段,分别插入原核表达载体,构建出重组菌BL21-pGEX-6p-1-lhp和BL21(DE3)-pET-30a(+)-lhp-linker-esat-6,经IPTG诱导表达得到融合蛋白。SDS-PAGE结果显示表达的融合蛋白GST-CFP-10和His-CFP-10-linker-ESAT-6与理论值大小相符,为进一步研究该蛋白的免疫学功能提供了条件。二、结核分枝杆菌CFP-10蛋白单克隆抗体的制备与鉴定应用淋巴细胞杂交瘤技术,以重组菌BL21(DE3)-pET-30a(+)-lhp表达的融合蛋白His-CFP-10为免疫原经纯化免疫8周龄BALB/c小鼠,皮下注射,100μg/只。首免时,抗原与等量弗氏完全佐剂混合乳化;二免、三免时抗原与等量不完全弗氏佐剂混合乳化,间隔时间为两周;以不加佐剂的抗原尾静脉加强免疫,100μg/只,3d后,取免疫鼠脾细胞和小鼠骨髓瘤细胞Sp2/0-Ag-14进行融合。以纯化的GST-CFP-10作为检测抗原,用间接ELISA方法筛选阳性克隆,获得7株稳定分泌CFP-10 McAb的细胞株,命名为2E7、6E8、7B11、7C2、7C3、7D4、8F6,腹水的效价依次为1×10~6、1×10~6、1×10~6、1×10~5、1×10~5、1×10~4、1×10~4。除6E8、7811 McAb亚类为IgG1外,其余均为IgG2b。Dot-ELISA试验表明,7株单抗只与表达His-CFP-10、GST-CFP-10及His-CFP-10-linker-ESAT-6的重组大肠杆菌反应,与其它菌株不反应。Western-blot试验显示7株单抗均能与融合蛋白His-CFP-10发生反应,出现特异性条带,而与BL21(DE3)-pET-30a(+)不反应。以上试验证实,筛选获得的McAb 2E7、6E8、7811、7C2、7C3、7D4、8F6是针对CFP-10的特异性单克隆抗体,它们为研究结核分枝杆菌CFP-10蛋白功能以及结核病的诊断提供了重要的条件。
[Abstract]:Tuberculosis (TB) is a chronic and consumptive infectious disease, which has become the number one killer and the biggest cause of death among infectious diseases. Tuberculosis, especially in developing countries, is especially severe among people infected with HIV. With the increase of the number of AIDS patients and the production of drug-resistant strains, the incidence of tuberculosis is increasing year by year. Therefore, for the prevalence of the disease, the early diagnosis of infection is very important. It is urgent to further improve the existing diagnostic methods. Lhp gene is a specific gene of Mycobacterium tuberculosis. Lhp is associated with the virulence of the bacteria, which can reduce the virulence of Mycobacterium tuberculosis. CFP-10 is the expressed protein of lhp. In this study, recombinant Escherichia coli expressing CFP-10 protein was constructed, and bioactive fusion protein was obtained, and CFP-10 specific monoclonal antibody was prepared. These results provide important conditions for studying the pathogenic mechanism and immune mechanism of Mycobacterium tuberculosis, and provide a new reagent for the diagnosis and quarantine of tuberculosis. Firstly, the prokaryotic expression of CFP-10 in the lhp gene of Mycobacterium tuberculosis is the dominant antigen, and its function can deepen the study on the pathogenic mechanism and immune mechanism of Mycobacterium tuberculosis. In this study, lhp gene and lhp-linker-esat-6 fragment were amplified by PCR and GeneSOEing, respectively, and inserted into prokaryotic expression vector respectively. The recombinant bacteria BL21-pGEX-6p-1-lhp and BL21 (DE3) -pET-30a () -lhp-linker-esat-6, were constructed. The expression of the fusion protein was induced by IPTG. The results of SDS-PAGE showed that the expressed fusion proteins GST-CFP-10 and His-CFP-10-linker-ESAT-6 were in agreement with the theoretical values, which provided the conditions for further study of the immunological function of the fusion protein. Preparation and Identification of Monoclonal Antibody against Mycobacterium Tuberculosis CFP-10 protein using Lymphocyte hybridoma technique, The fusion protein His-CFP-10 expressed by recombinant strain BL21 (DE3)-pET-30a ()-lhp was used as immunogen to immunize 8-week-old BALB/c mice subcutaneously, 100 渭 g / mouse. The antigens were emulsified with the same amount of Freund's complete adjuvant for the first time, and the antigens and the same amount of incomplete Freund's adjuvant were mixed emulsified for the second and third immunity respectively, with the interval of two weeks. The immunized mice were immunized with non-adjuvant antigenic tail vein, 100 渭 g / mouse, 3 days later, the spleen cells of immunized mice were fused with Sp2/0-Ag-14 of mouse myeloma cells. The purified GST-CFP-10 was used as the detection antigen, and the positive clones were screened by indirect ELISA method. Seven cell lines secreting CFP-10 McAb stably were obtained, named as 2E7E7, 6E8, 7B11, 7C2C3, 7D4, 8F6, and the titer of ascites was 1 脳 1061 脳 1061 脳 1061 脳 1061 脳 1051 脳 10 1 脳 10 5 1 脳 10 1 脳 10 4. The titer of ascitic fluid was 1 脳 10 1 脳 10 6 1 脳 10 6. The titer of ascitic fluid was 1 脳 10 6 1 脳 10 6, 1 脳 10 5, 1 脳 10 5, 1 脳 10 4. With the exception of 6E8O7811 McAb subclass being IgG1, the IgG2b.Dot-ELISA test showed that the seven McAbs reacted only with recombinant Escherichia coli expressing His-CFP-10,GST-CFP-10 and His-CFP-10-linker-ESAT-6. Western-blot test showed that all of the seven McAbs could react with the fusion protein His-CFP-10 with specific bands, but not with BL21 (DE3)-pET-30a (). The results showed that McAb _ 2E7N _ 6E _ 8C _ (78) 7C _ (2) C _ (2) C _ (7) C _ (2) C _ (3) C _ (7) D _ (4) F _ (6) was a specific monoclonal antibody against CFP-10, which provided important conditions for studying the function of CFP-10 protein of Mycobacterium tuberculosis and the diagnosis of tuberculosis.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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