TAT-Ag85B蛋白疫苗辅以T-bet佐剂抗结核效应研究

发布时间：2018-06-04 10:53

本文选题：结核分枝杆菌 + T-bet　；参考：《安徽理工大学》2014年硕士论文

【摘要】：目的通过原核表达TAT-Ag85B融合蛋白,与T-bet基因佐剂配伍构建新型抗结核病复合疫苗,评价新型疫苗的免疫原性及抗结核感染的保护作用,探究新型复合疫苗增强免疫应答的机制,为新型结核疫苗的研发提供新的思路和理论基础。方法 .1.根据GenBank报道的目的基因(Ag85B和TAT-PTD基因)全长序列,构建pET-28a-TAT-Ag85B和pET-28a-Ag85B重组质粒,经菌液PCR、质粒双酶切及测序鉴定。 2.将含有pET28a-TAT-Ag85B和pET-28a-Ag85B质粒的重组大肠杆菌经IPTG诱导、镍离子亲和层析纯化Ag85B、TAT-Ag85B蛋白,用Western blot法鉴定纯化后的蛋白。 3.将Ag85B、TAT-Ag85B蛋白和本实验室保存的pcDNA3.1-T-bet质粒免疫BALB/c小鼠,末次免疫结束后用ELISA法检测小鼠血清中抗Ag85B抗体滴度。同时将小鼠脾脏淋巴细胞于Ag85B或TAT-Ag85B刺激下培养,ELISA法检测培养液中细胞因子分泌情况。 4.将各组末次免疫结束后的小鼠经尾静脉注射感染结核分枝杆菌标准株H37Rv,在感染后第一、二、四、八周检测小鼠肺和脾内结核菌载量并在感染后八周取肺脏做组织病理切片。 5.用Ag85B、TAT-Ag85B蛋白分别刺激小鼠腹腔巨噬细胞,免疫荧光观察巨噬细胞内Ag85B蛋白的分布,Western blot检测细胞内Ag85B蛋白的含量,流式细胞术检测巨噬细胞表面分子CD80/CD86的表达。结果 1.成功构建pET-28a-TAT-Ag85B和pET-28a-Ag85B质粒,PCR鉴定、质粒双酶切鉴定和测序鉴定结果正确。 2.含有pET28a-TAT-Ag85B和pET-28a-Ag85B质粒的大肠杆菌经诱导、表达、纯化得到重组Ag85B.TAT-Ag85B蛋白,经Western blot鉴定为所需目的蛋白。 3.使用ELISA方法检测各组免疫小鼠血清中抗Ag85B抗体滴度情况,发现总IgG抗体效价中,TAT-Ag85B/T-bet组最高,高于其他实验组；TAT-Ag85B/T-bet与TAT-Ag85B、Ag85B组相比IgG2a抗体滴度明显增高(P0.05), IgG1滴度明显降低(P0.05)；从脾细胞分泌细胞因子水平来看,TAT-Ag85B/T-bet组小鼠IFN-γy/IL-2水平显著升高(P0.05), IL-4/IL-10水平低于Ag85B和TAT-Ag85B组(P0.05)。 4.H37Rv感染各组小鼠后,在第1周和第2周,各组小鼠的肺组织和脾组织结核菌载量无明显差异,在第4周TAT-Ag85B/T-bet组小鼠肺组织和脾组织结核菌载量明显低于PBS对照组(P0.05),在第8周时,TAT-Ag85B/T-bet、脾和肺组织结核菌载量低于对照组,有显著性差异(P0.05)；从肺组织大体外观判断,与对照组小鼠肺部外侧明显的结核灶分布相比,TAT-Ag85B/T-bet免疫组肺部外观几乎无肉眼可见结核结节；HE染色发现TAT-Ag85B/T-bet免疫组小鼠的肺组织中病变明显减轻,少量的充血、坏死和实变,有少量不明显的肉芽肿结节形成；抗酸染色结果显示在TAT-Ag85B/T-bet免疫的小鼠的肺组织中结核杆菌呈零星散在分布,无簇状或片状分布。 5.与Ag85B刺激巨噬细胞相比,TAT-Ag85B蛋白大量散在分布于巨噬细胞内,经TAT-Ag85B蛋白刺激后的巨噬细胞表面抗原提呈分子CD80/CD86表达荧光强度高于Ag85B蛋白。结论本研究初步阐明TAT-Ag85B蛋白疫苗辅以T-bet基因佐剂可以引起小鼠体内强烈的特异性免疫应答,并且以Thl优势免疫应答为主,对小鼠有一定的抗结核保护作用；TAT-Ag85B蛋白的跨膜转运功能可能在提高巨噬细胞抗原提呈能力中起重要作用,并参与了增强的抗结核免疫应答；T-bet佐剂对Thl优势免疫的诱导具有重要作用。总之,该复合型疫苗的研究将为新型结核疫苗的研发提供新的思路和理论基础。
[Abstract]:Purpose

Through prokaryotic expression TAT - Ag85B fusion protein , the novel anti - tuberculosis composite vaccine is combined with the T - bet gene adjuvant to evaluate the immunogenicity of the novel vaccine and the protective effect of anti - tuberculosis infection , and the mechanism for enhancing the immune response of the novel composite vaccine is explored , and a new thought and theoretical basis for the development of the novel tuberculosis vaccine is provided .

method

1 . The recombinant plasmids pET - 28a - TAT - Ag85B and pET - 28a - Ag85B were constructed according to the full - length sequence of the gene of interest ( Ag85B and TAT ) reported in GenBank . The recombinant plasmids of pET - 28a - TAT - Ag85B and pET - 28a - Ag85B were constructed .

2 . The recombinant E . coli containing pET28a - TAT - Ag85B and pET - 28a - Ag85B plasmid was induced by IPTG . Ag85B and TAT - Ag85B proteins were purified by nickel ion affinity chromatography . The purified protein was identified by Western blot .

3 . BALB / c mice were immunized with Ag85B , TAT - Ag85B protein and pcDNA3.1 - T - bet plasmid stored in this lab . After the last immunization , anti - Ag85B antibody titer was detected by ELISA . At the same time , mouse spleen lymphocytes were cultured under the stimulation of Ag85B or TAT - Ag85B , and the secretion of cytokines in the culture medium was detected by ELISA .

4 . After the final immunization of each group , the mice infected with Mycobacterium tuberculosis H37Rv were infected with Mycobacterium tuberculosis at the first , two , four and eight weeks after infection , and the lung and spleen of the mice were detected in eight weeks after infection , and the pathological sections of the lungs were taken after 8 weeks after infection .

5 . Ag85B and TAT - Ag85B protein were used to stimulate the distribution of Ag85B protein in macrophages . Western blot was used to detect the expression of Ag85B protein in macrophages . Western blot was used to detect the expression of Ag85B protein in macrophages .

Results

1 . pET - 28a - TAT - Ag85B and pET - 28a - Ag85B plasmid were constructed successfully , and the results were identified by PCR .

2 . E . coli containing pET28a - TAT - Ag85B and pET - 28a - Ag85B plasmid was induced , expressed and purified to obtain recombinant Ag85B . TAT - Ag85B protein , which was identified by Western blot as the desired protein .

3 . ELISA was used to detect the titer of anti - Ag85B antibody in serum of each group , and found that TAT - Ag85B / T - bet group was the highest in the total IgG antibody titer , which was higher than that of other experimental groups .
Compared with TAT - Ag85B , Ag85B , TAT - Ag85B / T - bet was significantly higher than that of TAT - Ag85B and Ag85B groups ( P0.05 ) .
The levels of IFN - 纬y / IL - 2 in TAT - Ag85B / T - bet group were significantly higher than that in TAT - Ag85B / T - bet group ( P0.05 ) . IL - 4 / IL - 10 levels were lower than that in Ag85B and TAT - Ag85B groups ( P0.05 ) .

4 . After infected with H37Rv , there was no significant difference between lung and spleen in groups 1 and 2 , and the amount of TB bacteria in lung and spleen in TAT - Ag85B / T - bet group was significantly lower than that in control group ( P0.05 ) . At 8th week , TAT - Ag85B / T - bet , spleen and lung tissue TB were significantly lower than that in the control group ( P0.05 ) .
Compared with the control group , the lung appearance of TAT - Ag85B / T - bet immune group was almost free from nodule nodules .
HE staining showed that in TAT - Ag85B / T - bet immune group , the pathological changes of lung tissue were obviously alleviated , and little congestion , necrosis and change were found , and there were few obvious granulomas nodule formation .
The results showed that Mycobacterium tuberculosis in the lung tissues of TAT - Ag85B / T - bet - immunized mice was sporadic in sporadic , non - clustered or sheet - like distribution .

5 . Compared with Ag85B stimulated macrophages , TAT - Ag85B protein was distributed in macrophages , and the fluorescence intensity of the surface antigen of macrophages stimulated by TAT - Ag85B protein was higher than that of Ag85B protein .

Conclusion

The results of this study indicate that TAT - Ag85B protein vaccine can induce a strong specific immune response in mice by using the T - bet gene adjuvant , and has a certain anti - tuberculosis protective effect on mice with Thl dominant immune response .
The transmembrane transport function of TAT - Ag85B protein may play an important role in increasing the antigen presentation ability of macrophages and participate in the enhanced anti - tuberculosis immune response .
T - bet adjuvant plays an important role in the induction of Thl dominant immune . In conclusion , the research of the compound vaccine will provide new ideas and theoretical bases for the research and development of new tuberculosis vaccines .
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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