【作者】 任永明；

【导师】 罗耀武； 黄华樑；

【作者基本信息】 河北农业大学 ， 作物遗传育种学， 2002， 硕士

【摘要】 大肠杆菌是最常用的外源蛋白表达宿主，它以其遗传背景清楚，便于操作和成本低廉而在基因工程中广泛使用。但是在基因工程的实践中，大量表达的外源蛋白往往容易聚集成难溶的包涵体。这就需要复杂的变性复性过程，使其从无活性的聚集体状态恢复成为天然构象状态。同时人们发现在极端渗透压环境中，细菌能够通过摄取或者合成有限的几种所谓的相容性溶质（compatible solute）而保护自己。利用这一特性，可以使用相容性溶质来解决重组蛋白的不溶性问题。在本实验中，我们首先从抗膀胱癌单链抗体表达载体pWAI80-Pg和免疫毒素构建的中间载体pMS-38-T（编码 PE38）构建了抗膀胱癌免疫毒素表达载体pABDIT，其结构为：pelB-V_H-linker-V_L-HiS₅-connector-PE38。pelB为细菌果胶酶信号肽，可将表达产物引导到周质腔。His₅标签便于用金属螯合层析纯化。较长的connector（15个氨基酸）保证单链抗体和毒素独立的进行折叠。在此基础上，用重叠PCR法在免疫毒素羧基端C-Myc标签序列和用KDEL序列代替RDELK序列。C-Myc标签可以使检验更加方便，而KDEL则可以进一步增加免疫毒素的细胞毒作用。在整个免疫毒素中scFv代替了完整的毒素分子的细胞结合结构域，特异性的靶向膀胱癌细胞，而绿脓杆菌外毒素A的截断产物PE38则发挥细胞毒作用。采用不同的培养和诱导条件，使免疫毒素以包涵体和可溶两种形式表达。包涵体经过复性和离子交换层析得到纯化。可溶表达的免疫毒素经过一次亲和层析得到纯化。免疫毒素的特异性经过western blotting鉴定。据调查，本研究中所采用的利用相容性溶质在高盐渗透压胁迫下表达重组蛋白为国内首次报道，在基因工程实践中为解决包涵体问题摸索了一条可供选择的新路；同时在实验中发现葡萄糖对基因表达调控的影响具有一定的理论价值。 因为植物表达体系有：成本低、与动物细胞相似的真核表达环境、总体产量高等诸多优点，人们越来越多的把目光投向具有商业价值的转基因植物。本文尝试以抗膀胱癌免疫毒素为目的基因，构建植物表达载体，转化烟草，研究免疫毒素在植物中的表达。构建成的植物表达载体pBBDIT构建的载体以pABDIT2为基础，经过酶切位点的改造，连入改造后的植物表达载体质粒pBNVPDE中，其结构为：Nos-pro-NPTII-Bi-35S-PelB-ScFv-PE38mycKDEL-Nos-ter，与大肠杆菌表达载体一样，可以预期整个表达产物以pelB为信号肽，利用单链抗体的靶向作用和PE38的杀伤作用，可以实现对特定靶细胞的杀伤。C-Myc标签可以使检验更加方便，而KDEL则可以进一步增加免疫毒素的细胞毒作用。然后利用电激法将其转入农杆菌LBA4404中，叶盘法转染烟草，卡那霉素筛选得转化苗。经PCR进一步筛选和鉴定，得到转基因植株，取2株作Western blotting验证了表达产物的特异性。据调查，本研究为国内外首次利用转基因植物表达免疫毒素，具有一定的理论价值，同时也为大规模低成本生产靶向药物分子积累了经验。更多还原

【Abstract】 The Gram-negative bacterium Escherichia Coli remains the most versatile host for the production of heterologous protein. Although for most applications it is desirable to achieve maximal production within the cytoplasm, the inclusion body is a major difficulty in production of heterologous proteins. Thus we need ardously denature and renature the inclusion bodies. However, another method has emerged which based on the following fact: bacterial protect themselves against high external osmolality by uptake or synthesis of a limited number of so-called compatible solutes. Under such extreme conditions. combine factors can facilitate the foreign recombinant protein expression. \Ve constructed expression vector pABDIT2 ,which was composed of the scFv derieved from anti-human bladder carcinoma monoclonal antibody BDI(scFv) and a truncated from Pseudomonas exotoxin(PE38).In this construct, scFv serves as the binding domain and the toxin fragment as the functional domain. The signal sequence pelB directs the transportation of the product to periplasm, and long connector between the scFv and PE38 allow them to fold independently and thus correctly . To facilitate purification of the recombinant protein, we introduced an HIS5 tag into this vector. The C-myc tag and four amino acids KDEL also were added to the C terminal of the fusion protein using SoE-PCR. Under different growth and induction conditions, the imunotoxin was expressed in two forms. The insoluble form, inclusion body, which was cultured and induced in the normal condition was renatured and purified using anion-exchange chromatography. In the alternative condition, we add additional NaCl and two compatible solutes (glycine betaine and sorbitol) into the medium. Then the recombinant immunotoxin BDI(scFv)-PE38 mycKDEL was expressed mainly in soluble form and can be purified by Ni2+-NTA agarose. We also found the time when the glucose was added into the medium will greatly affect the expression level of the recombinant immunotoxin. This suggests that glucose may play an important role in the expression system.The production of recombinant proteins in plants has many potential advantages for58generating biopharmaceuticals relevant to clinical medicine : more economical , fewer purification requirement, similar posttranslational modification with animal cell than bacterial and so on. So .the plant expression system has became a promising one.In this experiment, the gene of BDI(scFv)-PE38KDEL against human bladder carcinoma was chosen as objective gene to construct vector for expressing in plant and transform tobacco, so as to research the expression of engineering antibody in plants. After the plasmid pBABIT had been constructed, it was transferred to Agrobacterium tumefacient LB4404 by electrotranstormation was carried out by leaf disc method. Selected and identified by PCR, several transgenic plants has been obtained. The specific bands of western blotting have shown that the recombinant immunotoxin was expressed in tobacco successfully.更多还原