TNFα自体疫苗的制备、筛选及其对TNFα表达过高相关疾病治疗作用的研究

发布时间：2018-08-23 15:41

【摘要】： 研究背景TNFα是一种具有复杂生物活性的细胞因子,它主要由巨噬细胞和T细胞产生。该分子除了能够在体内或体外杀伤肿瘤细胞外,还具有抗病毒感染、激活巨噬细胞及多形核粒细胞、促进组织相容性抗原表达、调节机体免疫机能等多种功能。但是当肿瘤坏死因子过量表达时,它与其它炎症因子一起产生多种病理损伤,如自身免疫性疾病,感染性疾病,恶液质等。因此,中和体内过量的TNFα,可为上述疾病的治疗提供一条有效途径。目前临床上通过中和体内过量的TNFα治疗上述疾病的药物有:TNFα单克隆抗体和TNFα可溶性受体,但是这些药物存在用药量大、需长期反复使用、易引起超敏反应的缺陷。疫苗只需少量注射就可以诱发产生抗体,可克服抗体及可溶性受体药物的缺陷,因此TNFα疫苗是治疗此类疾病的一条有效途径。目的本研究的目的是筛选出一种TNFα自体疫苗分子,使之能够在体内诱导出针对TNFα分子的特异性中和抗体,为TNFα表达过高的相关疾病提供一种新的治疗药物。方法构建GST融合的小鼠TNFα(mTNFα)系列截短体,使其在大肠杆菌中获得高效表达,菌体蛋白经SDS-PAGE后,切胶免疫小鼠,通过对诱导抗体滴度的分析并结合计算机辅助设计确定外源T细胞辅助表位的插入位置。将编码TT830-844(QYIKANSKFIGITEL), HEL46-61(NTDGSTDYGILQINSR)和PADRE (AKFVAAWTLKA)氨基酸的基因序列替换编码小鼠TNFα126位至140位氨基酸序列的核酸,获得编码mTNF-TT,mTNF-HEL,mTNF-PADRE基因片段,将其插入pBV220原核表达载体,转化大肠杆菌,在大肠杆菌中经表达、纯化后得到mTNF-TT,mTNF-HEL,mTNF-PADRE融合蛋白,构建出三种TNFα融合蛋白疫苗。用构建的三种疫苗分子免疫小鼠,体外比较免疫的抗血清针对mTNFα的抗体滴度和中和活性,筛选最有效的T细胞辅助表位。用筛选得到的mTNF-PADRE治疗LPS诱导的内毒素休克小鼠,mTNFα诱导的恶病质小鼠以及Ⅱ型胶原诱导的关节炎(CIA)小鼠,观察治疗效果。结果通过对小鼠TNFα系列截短体诱发的抗mTNFα抗血清滴度检测和对人和小鼠TNFα抗原表位预测,结果发现小鼠TNFα126位至140位氨基酸可以作为外源T辅助表位替换的位置。我们用外源的T辅助表位替换小鼠TNFα126-140位氨基酸,构建了mTNF-TT,mTNF-HEL,mTNF-PADRE三种TNFα融合蛋白疫苗,经纯化纯度均可达到95%以上。L929细胞的杀伤活性试验结果表明,mTNF-TT,mTNF-HEL,mTNF-PADRE均不具有TNFα的细胞杀伤活性。免疫小鼠结果发现mTNF-TT,mTNF-HEL,mTNF-PADRE均可在小鼠体内诱导出小鼠TNFα结合性和中和性自身抗体,比较其效果,mTNF-PADRE具有最好的免疫效果。无佐剂的mTNF-PADRE免疫小鼠可以有效地延长LPS诱导的内毒素休克小鼠的生存期,减少mTNFα诱导的恶病质小鼠的体重减轻的幅度以及减轻Ⅱ型胶原诱导的关节炎模型(CIA)小鼠的症状。结论通过对表位插入位置和插入的表位的筛选,我们筛选的mTNF-PADRE分子可以有效地诱导出针对mTNFα的中和性自身抗体,并且不具有TNFα的L929细胞杀伤活性。动物试验表明mTNF-PADRE在无佐剂的情况下可以有效地缓解LPS诱导的内毒素休克,mTNFα诱导的恶病质以及CIA小鼠的症状。因此其对应的人TNF-PADRE分子可以作为TNFα自体疫苗的候选分子。
[Abstract]:Background TNFalpha is a cytokine with complex biological activity, which is mainly produced by macrophages and T cells. In addition to killing tumor cells in vivo or in vitro, TNFalpha also has many functions, such as anti-virus infection, activation of macrophages and polymorphonuclear granulocytes, promotion of histocompatibility antigen expression and regulation of immune function. However, when tumor necrosis factor is overexpressed, it produces a variety of pathological damage with other inflammatory factors, such as autoimmune diseases, infectious diseases, cachexia and so on. Therefore, neutralizing excessive TNF-a in vivo can provide an effective way for the treatment of these diseases. Drugs for these diseases include monoclonal antibodies against TNFalpha and soluble receptors for TNFalpha. However, these drugs have the disadvantages of large dosage, long-term repeated use and easy to cause hypersensitivity. OBJECTIVE To screen an autologous vaccine molecule of TNFalpha so as to induce a specific neutralizing antibody against TNFalpha molecule in vivo, and to provide a new therapeutic drug for the related diseases with high expression of TNFalpha. High expression was obtained in E. coli. After SDS-PAGE, mice were immunized by gel cutting. The insertion site of foreign T cell helper epitopes was determined by titer analysis and computer-aided design. TT830-844 (QYIKANSKFIGITEL), HEL46-61 (NTDGSTDYGILQINSR) and PADRE (AKFVAAWTLKA) amino acids were sequenced. The mTNF-TT, mTNF-HEL, mTNF-PADRE gene fragments were obtained by substituting the nucleic acids encoding murine TNF alpha 126 to 140 amino acid sequences. The fragments were inserted into the pBV220 prokaryotic expression vector and transformed into E. coli. After being expressed in E. coli, mTNF-TT, mTNF-HEL and mTNF-PADRE fusion proteins were purified. Three kinds of TNF-alpha fusion protein vaccines were constructed. Three vaccines were constructed to immunize mice. The titers and neutralizing activities of the antibodies against mTNF-alpha were compared in vitro to screen the most effective T-helper epitopes. The mTNF-PADRE was used to treat LPS-induced endotoxic shock mice, mTNF-alpha-induced cachexia mice and collagen-induced arthritis (CIA) mice. Results By detecting the titers of anti-mTNF-alpha antiserum induced by murine TNF-alpha truncated bodies and predicting the epitopes of human and murine TNF-alpha antigens, it was found that the amino acids at positions 126 to 140 of murine TNF-alpha could be used as the substitution sites for exogenous T-helper epitopes. We used exogenous T-helper epitopes to replace the amino acids at sites 126 to 140 of murine TNF-alpha. Three kinds of TNF-alpha fusion protein vaccines, mTNF-TT, mTNF-HEL and mTNF-PADRE, were constructed. The purity of these vaccines was above 95%. The cytotoxicity test of L929 cells showed that mTNF-TT, mTNF-HEL and mTNF-PADRE did not possess the cytotoxicity of TNF-alpha. Immune mice showed that mTNF-TT, mTNF-HEL and mTNF-PADRE could be induced in vivo. Mice immunized with mTNF-PADRE without adjuvant could prolong the survival time of LPS-induced endotoxic shock mice, reduce the weight loss of mTNF-alpha-induced cachexia mice and reduce the joint induced by collagen type II. Conclusion The mTNF-PADRE molecule can effectively induce neutralizing autoantibodies against mTNF-alpha and has no cytotoxic activity against TNF-alpha L929 cells. Animal experiments show that mTNF-PADRE can effectively alleviate the symptoms without adjuvant. To understand LPS-induced endotoxic shock, mTNF-alpha-induced cachexia and CIA mice symptoms, the corresponding human TNF-PADRE molecule can be used as a candidate molecule for TNF-alpha autoimmune vaccine.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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