Studies On Expression And Activity Of Recombinant Human Anti-HER2 Antibody In Pichia Pastoris

With the development of gene recombina nt technology, human had made greatimprovements on applied performa nce of antibodys from varies aspects and hadproduced a variety of gene engneered antibodies, such as, huma nized mousemonoclona l antibody, sma ll molecule antibodies, antibody fusion protein, etc. Theygreatly extended the scope of antibody's applica tion, lead the antibody projectdevelopment into a new period -- huma nized antibody period . The ma inly researchhotspots are concentrated on two parts : The huma niza tion of antibody and theimproving of huma nized antibody's affinity. So it is important to establish a expressionvector to screen and express different kinds of critica l genes, during the screeningprogress or enhancing the origina l antibody's affinity and quality of being neutralizedby taking the mutation or other technologies. With the development of proteinengineering technology, the structure of protein has been increasingly significa ntimpacting on fuction and activity of protein. The using of computer technology toanalysis the antibody's structure for improving uction and activity of antibody has beenanother research focus,through the technology of gene directional transformation, ect.So the research is intend to use the gene recombina nt technology, constructed ageneral vector based on the expression plasmid which named full huma nized anti-Rabies virus antibody, and Modified the cloning site for VH CDR3 freely changing,screening and expressing, by PCR med iated site-specific mutations. Constructed theanti-HER2 antibody expression plasmid by inserting of synthesized oligos: humanCDR3 and mouse CDR3, established the expression system in Pichia Pastoris.Throughanalysis the expression products in Pichia Pastoris to identify the recombina nt anti-HER2 antibody and evaluate the advantage of the general vector. The research also use the network database to compare the differences of the prima ry structure, secondarystructure and 3-D structure among the herceptin, recombina nt huma nized anti-HER2antibody and recombina nt murine anti-HER2 antibody. The comparison can find outthe key point which decide the activity of antibody and offer the theoretica la ndexperimental foundation of the exploitating and development HER2 monocloneantibody.The research have two parts:The first part: The construction of recombinant vector for expressing andscreening of humanized antibody and the expressing of recombinant humanizedand murine anti-HER2 antibody in Pichia PastorisBy PCR med iated site-specific mutations , we constructed a general vector basedon the expression plasmid which named full huma nized anti-Rabies virus antibody, andModified the cloning site for VH CDR3 freely changing, screening and expressing.Then we connect the synthesized oligos of human CDR3 and mouse CDR3 to thegeneral vector and established the expression system in Pichia Pastoris.Throughanalysis the expression products in Pichia Pastoris to identify the recombina nt anti-HER2 antibody and evaluate the advantage of the general vector.The experiment turns out that the general expressing vector pPICZαH3 wascreated successfully. Integration efficiency of the improved vector was increasedsignifica ntly. So besides full human antibody, the improved vector could also expressother multimers by VH CDR3's free switchover. Cloned the CDR3 into expressionvector pPICZαH3, analyzed the recombina nt protein by ELISA and Western blot aftertransformation. The results showed : the origina l huma n-anti-Rabies virus antibodyowns the ability as carcinoma of anti-breast HER2 antibody does, and the ability ofnurine antibody is higher than the huma nized antibody. From above all, we proved thatthe general vector pPICZαH3 not only can be used as the screening and expressingsystem for the functiona l antibody, but also mea ns a lot to the progress of antibody'shuma nized and the affinity maturation.The second part: The structure prediction and functional analysis ofrecombinant humanized and murine anti-HER2 antibody We get the herceptin protein sequence and HER2 antigen protein sequence fromthe NCBI datebase, and analysis the homology among the herceptin, recombina nthuma nized and murine anti-HER2 antibody. We also compare their protein physicaland chemica l properties and hydrophobic through the database http: // us. expasy.Org / tools. We use the SWISS-MODEL simulate the 3-D structures of recombina nthuma nized and murine anti-HER2 antibody, and compare the structures with 3-Dstructure of herceptin.The experiment results have demonstrated that the ma in differences amongHerceptin, recombina nt huma nized anti-hert2 antibody and recombina nt murine antihert2antibody existed in CDR. The three antibodies had 95 percent homology , 84percent between herc eptin and recombina nt huma nized anti-hert2 antibody and 85percent between herceptin and recombina nt murine anti-hert2 antibody. The physicochemical properties of them had no conspicuous differences and the isoelectric pointsand molecular weight of Herceptin, recombina nt huma nized anti-hert2 antibody andrecombina nt murine anti-hert2 antibody were 8.83,8.82,8.61 and 23404.24,23336.20,23363.22. The coefficients of stability of them which were instable proteinswere 40.32,42.28,40.18. With no difference in FR, different amino acids compositionma inly existed in CDR and the difference of hydrophobicity existed in CDR3. Thethree-dimensiona l structures binding to antigen of HER2 of the three proteins werecontrasted by the method of SWISS-MODEL, and the ma in differences existed inCDR3 where antibody and antigen binded.The functions of recombina nt huma nized and murine anti-HER2 antibodywere analyzed through biology and immunology and its results proved that therecombina nt vector could screen and express huma nize antibody and was successfullyconstructed. This was very important to make high affinity huma nized antibody and italso proved that the Pichia.pastoris expression system pla yed an important role incloning, screening, expressing interest antibodies and improving therapeuticmonoclona l antibody. With assista nt computer analysis, CDR3 could determine thethree-dimensiona l structure and activity of antibody and the studies of CDR3 were critica l to develop and improve the therapeutic monoclona l antibodies.