【作者】 张琴；

【导师】 李虹；

【作者基本信息】 四川大学 ， 病原生物学， 2007， 硕士

【摘要】 目的：铜绿假单胞菌外毒素(Pseudomonas Exotoxin A，PE)是铜绿假单胞菌(Pseudomonas aeuriginosa，PA)分泌的一种致病物质。PE为一条单链多肽，能抑制真核细胞的蛋白质合成，导致细胞死亡。自上世纪80年代中期以来，PE及其它一些植物的或细菌的毒素被用来与抗体、细胞因子、多肽类激素等偶联，而成为免疫毒素(Immunotoxins，ITs)，用以杀伤带有相应抗原或受体的靶细胞。目前已有多种PE类免疫毒素进入临床试验阶段，尤其在肿瘤治疗方面取得了显著的疗效。但是在动物实验及临床试验中，ITs亦表现出非特异性毒性，如导致发热、寒颤、血管渗漏综合症、肝肾损伤等毒性反应。普遍认为毒素分子本身和作为导向分子的抗体或者细胞因子都有可能是引起非特异性毒性反应的因素。故本研究拟构建截短的PE(PE38KDEL)的原核表达载体，进行目的蛋白的表达和纯化，并检测其对细胞的毒性活性，从而为进一步改造毒素，降低非特异性毒性作用，为构建更加有效的免疫毒素提供高效低毒的毒素分子奠定实验基础。方法：(1) PE38KDEL的原核表达载体的构建：从GenBank上获取PE38KDEL基因序列，使用引物设计软件Primer Premier 5．0进行引物设计，再利用PCR技术从原核表达质粒pRKL／IL18-PE38KDEL扩增目的基因片段PE38KDEL。通过酶切及连接反应将PE38KDEL克隆入原核表达载体pGEX-4T-1，构建原核表达质粒pGEX-4T-1／PE38KDEL。重组质粒经过限制性内切酶酶切鉴定、PCR扩增鉴定及DNA序列测定证实插入片段的正确性。(2)GST-PE38KDEL融合蛋白在大肠杆菌BL21(DE3)中的表达鉴定及活性测定：将鉴定正确的阳性重组质粒pGEX-4T-1／PE38KDEL转化大肠杆菌BL21(DE3)，IPTG诱导重组质粒的表达，表达产物经SDS-PAGE电泳及Western blot分别测定其大小和特异性。用GSTrap FF纯化柱纯化融合蛋白GST-PE38KDEL。采用MTS比色法检测融合蛋白对细胞的杀伤作用。结果：(1)经限制性内切酶酶切鉴定、RCR扩增及序列测定证实，截短的铜绿假单胞菌外毒素(PE38KDEL)的原核表达质粒构建成功，目的片段大小正确，定向插入的方向正确，目的基因的阅读框架无改变。(2)重组质粒pGEX-4T-1／PE38KDEL在大肠杆菌BL21(DE3)中获得了PE38KDEL与GST的融合表达，SDS-PAGE结果显示表达产物的分子量大小与预期值一致，Western blot结果显示表达产物可与兔抗PE多克隆抗体结合，在NC膜上显示出一条与预期分子量一致的蛋白印迹条带。上清可溶性蛋白经GSTrap FF纯化柱纯化，纯化产物行SDS-PAGE检测，结果显示获得单一的Mr约63 000的目的条带。MTS比色法检测细胞毒性实验，结果显示目的蛋白在作用浓度1～10μg／ml时对实验组细胞没有或有轻微的杀伤作用，在100～1000μg／ml杀伤作用显著。结论：(1)成功构建了截短的铜绿假单胞菌外毒素(PE38KDEL)的原核表达载体—pGEX-4T-1／PE38KDEL。(2)pGEX-4T-1／PE38KDEL在大肠杆菌BL21(DE3)中获得了高效的表达，经纯化得到了纯度较高的融合蛋白。MTS比色法检测结果显示，融合蛋白GST-PE38KDEL在高浓度下对多种细胞具有强烈的杀伤作用。本研究结果为下一步改造毒素，降低其非特异性毒性，以构建高效低毒的免疫毒素奠定了基础。更多还原

【Abstract】 Objective:Pseudomonas Exotoxin A (PE) is one of the pathogenic proteins that is secreted by Pseudomonas aeuriginosa. It helps the bacteria invade body tissues. PE is composed of a single peptide chain, and inhibits protein synthesis in eukaryotic cells and causes cell death. Since 1980s, several plant toxins and bacterial toxins have been coupled to antibodies and cytokines to make immunotoxins specific for surface properties in order to kill target cells specifically. These immunotoxins are now being applied for treatment of cancers, autoimmune diseases, and chronic infectious diseases, especially for the treatment of malignant tumor. At present, many researches of imminotoxins containing PE have reached the stage of clinical trials. Some trials of these immunotoxins targeting tumors are currently in progress. However, unexpected toxicities appears to produce intolerable side effects, for example, fever, chill, vascular leak syndrome (VSL), injury of liver or kidney.In this study, the recombinant plasmid pGEX-4T-1/PE38KDEL was constructed, and transformed into E. Coli BL21(DE3) by chemical transformation. The fusion protein GST-PE38KDEL was expressed in E. Coli after induced by IPTG, and purified by GSTrap FF column. The cytotoxicity of the fusion protein was detected by MTS assay in vitro. These results provided some useful data to mutate PE38KDEL residues to ameliorate non-specific toxic and construct more effective immunotoxins with modified PE38KDEL.Methods:(1) The construction of prokaryotic expression vector by using truncated Pseudomonas Exotoxin A (PE38KDEL): we acquired PE38KDEL gene sequence from GenBank. According to the sequence, the primers for amplifying PE38KDEL gene were designed by software Primer Primier 5.0. PE38KDEL gene was amplified by PCR from the plasmid pRKL/IL18-PE38KDEL. PCR products were cloned into prokaryote expression vector pGEX-4T-l after being digested by EcoRⅠand NotⅠto construct the recombinant plasmid pGEX-4T-1/PE38KDEL. The recombinant plasmid was identified by restriction endonucleases digestion, PCR and sequence analysis.(2) The expression and characteristics of fusion protein PE38KDEL with GST in E. colt After the pGEX-4T-1/PE38KDEL transformed into E. coli BL21(DE3) and induced by IPTG, the expression product was obtained. The molecular weight and specificity of the fusion protien were identified by SDS-PAGE and Western blot. The fusion proteins GST-PE38KDEL with GST was purified through the GSTrap FF column. The cytotoxicity of fusion protein GST-PE38KDEL was checked by MTS assay in vitro.Results(1) After verification of the cloning fidelity by restriction endonucleases digestion, PCR, and sequencing, the successful constructions of prokaryote expression plasmids pGEX-4T-l/PE38KDEL were confirmed. The sequence of PE38KDEL was identical to that of PE in GenBank and the ORF was also not changed.(2) The BL21(DE3) transformed with pGEX-4T-I /PE38KDEL expressed fusion protein GST-PE38KDEL. The molecular weight of expression product was identical to the expected size, and showed the fusion protein with Mr of 63 000. The expression product was able to react with the specific anti-PE rabbit sera when it was detected by Western blot. The soluble fusion protein was purified by GSTrap FF column. The protein of Mr 63 000 was obtained when the GSTrap FF column was washed with 10 mmol/L Elution buffer. The cytotoxicity of fusion protein was detected by MTS assay. The result indicated that it was none or slightly toxic in several cell lines by the addition of the fusion protein GST-PE38KDEL to a final concentration of 1～10 ug/ml, but the fusion protein GST-PE38KDEL had extremely cytotoxicity at 100～1000μg/ml.Conclusion(1) The prokaryote expression vectors coding truncated Pseudomonas Exotoxin A (PE38KDEL) were constructed successfully.(2) After the recombinant plasmid pGEX-4T-l/PE38KDEL transformed into E. coli BL21(DE3), large amounts of fusion protein GST-PE38KDEL were purified. By cytotoxicity assay, The target protein was extremely toxic in several cell lines at high concentration. The results provided some useful data to mutate PE38KDEL residues to ameliorate non-specific toxic and construct more effective immunotoxins with modified PE38KDEL.更多还原