【作者】 秦兰霞；

【导师】 赵建军；

【作者基本信息】 中国人民解放军军需大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 绿脓杆菌外毒素A(Pseudomonas Exotoxin A,PEA)是绿脓杆菌的主要毒力因子之一，其毒性作用必须在毒素分子进入细胞后才发挥出来。PEA为一条单链毒蛋白，具有三个功能区，分别为功能区Ⅰa，功能区Ⅰb，功能区Ⅱ和功能区Ⅲ。功能区Ⅰa负责识别和结合细胞，功能区Ⅱ负责跨膜移位，功能区Ⅲ具有ADP-核糖基化活性，可通过对延伸因子EF-2的ADP-核糖基化作用而抑制真核细胞的蛋白合成。人组蛋白H3是碱性核蛋白，富含精氨酸，在生理条件下，H3的精氨酸带正电荷，而DNA的磷酸基团带负电荷，所以组蛋白和DNA分子主要依靠静电引力相结合。 本实验在已成功获得的PEA功能区Ⅰa，功能区Ⅱ基因片段与人组蛋白H3基因片段的克隆菌株的基础上，构建原核表达载体，将克隆质粒pMD-18T-PEA经Nco Ⅰ，Mlu Ⅰ双酶切，回收酶切产物；将克隆质粒pMD-18T-H3经Mlu Ⅰ，Xho Ⅰ双酶切，回收酶切产物。同时将原核表达载体pET-28c用Nco Ⅰ，Xho Ⅰ双酶切，回收酶切产物，将回收的酶切产物PEA，H3，载体进行连接，并转入DH5α感受态细胞内，培养12-18小时后，挑取阳性菌落，经Nco Ⅰ，Xho Ⅰ双酶切分析及PCR检测，筛选到阳性克隆，其质粒测序结果表明成功地构建了毒性基因缺失的PEA与人组蛋白H3融合基因的原核表达载体。 提取重组质粒并转化大肠杆菌BL21(DE3)，经IPTG诱导阳性菌，对菌体总蛋白进行SDS-PAGE电泳分析，结果表明BL21(DE3)能有效表达目的蛋白，薄层扫描计算分析，显示表达蛋白带密度积分占总蛋白量的16.28％。大量诱导培养表达菌，制备包涵体，经侧带法纯化后加入弗氏佐剂制成免疫原，对獭兔进行3次(每次间隔14天)注射免疫，通过ELISA法测定，该重组蛋白能够诱导獭兔产生抗体，免疫后7-14天达到峰值，并初步制备了终点滴度为1：1600的兔抗PEA-H3抗血清。大量制备包涵体，并对包涵体制备方法优化。利用融合蛋白带有6个组氨酸标签的特点采用金属螯和亲和层析对变性溶解的融合蛋白进行纯化，纯化结果含有少量杂带。用鲑鱼精DNA和质粒与表达蛋白作用，进行表达蛋白与DNA的凝胶迁移率变动分析实验，证明了融合蛋白与DNA的结合效果非常好。采用溴化氰活化的Sepharose 4 Fast Flow进行亲和层析。用聚乙二醇（PEG）10000浓缩，G－250法测含量，计算蛋白产率为30％。表明亲和层析纯化效果非常理想。从而为进一步的重组蛋白功能分析和基因转染，基因治疗实验奠定了基础。更多还原

【Abstract】 Pseudomonas exotoxin A (PEA) is a single chain toxin,PEA is made up of three domains. The pHysical boundaries indicated that domain Ia is composed of residues l-252;domain II, residues 253-364; domainIb, residues 365-404; and domain III, residues 405-613. It has been shown that domain Ia is responsible for cell recognition, domain II is to be involved in translocation of the toxin across membranes,and domain HI catalyzes the ADP-ribosylation of elongation factor2, Which arrests protein synthesis and results in cell death. Arginine rich human histone H3 is a kind of basic nucleosome protein.In the medium of pHysiological condition,by electrostatic interaction,histone H3 which arginine impart to it a positive charge binds to DNA which pHospHate groups impart to it a negative charge.In this paper,In order to establish a new technology of transfection, the functional domains la and II gene segments of PEA were lined with histone H3 gene segment, then a recombinant fusion protein was constructed which serves as carrier for transfer of DNA via receptor-mediated endocytosis.In this study,the recombinant plasmid pMD-18T-PEA-H3 was cleavaged with NcoI ,XhoI and inserted into the expression vector pET-28c and subsequently subjected to restriction endonuclease analysis and sequencing, the result indicated that the prokaryotic expression vector pET-28c-PEA-H3 was constructed successfully.After the expression plasmid was extracted and transformed into expression hosts BL21(DE3) of E.coli,the transformed hosts were induced by IPTG,bySDS-PAGE and ELISA analysis of host protein.the expression of theobjective gene was detected ,and it could account for 16.28% of the total host protein .inclusion body was prepared from the incubating expression hosts induced by IPTG. After purification and absolv in 8mol/L urea .And then injected rabbits 3 times with 14 days interval.By analysis of indirect ELISA, the anti-PEA-H3 antibody appear in the rabbit serum induced by the recombinant fusion protein,the highest antibody level was observed during 7-14days after the final inoculation .Incubate the genetic engineering bacteria in LB medium(Kana) and induce the inclusion body with IPTG ,then sonicated the bacterial cells and then subjected to denaturation in 8mol/L urea.Then purified inclusion body with His trap?metal affinity chromatograpHy.In order to obtain higher purity the expressed protein.Protein was further purified by CNBr-activated SepHarose 4 Fast Flow with nonspecial affinity chromatograpHy .At last,PEA-H3protein eventually collect 30%,it was prepared for next experiments functional analysis of the fusion proteinand genetic transfaction.更多还原