Expression Of Single-chain Immuonotoxin Against Human Bladder Carcinoma In Escherichia Coli And Tobacco

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.