| Genetically engineered antibody is third generation antibody after the multiclone antibody and the monoclone antibody. The genetically engineered antibody can not only reserve or increase the speciality and bio-activity of the natural antibody, but also dispose or decrease the unrelated stucture, degrade or eliminate the immunity of the antibody, making the antibody humanized, and improving the pharmacokinetics of the antibody. Moreover, it can be produced easily and is inexpensive. We can gain the rare antibody easily in this way, and it has a widely clinical application prospect. The fundamental principle of the technology is: firstly, extract mRNA from hybridoma cells,immuned splenic cells or peripheral blood lymphocyte,use reverse transcription to get cDNA. Secondly, expand the gene of the heavy chain and the light chain of antibody respectively through PCR, then connect the two pieces of genes and clone the product in the expression vector in a certain way. At last, express it in appropriate host cell(e.g. Escherichia coli, CHO cells, yeast cells, vegetal cell and insect cells) and fold the protein we get into functional antibody. Filtrate high-effected cell strain.According to the principle above,by using the technology of gene recombination,we choose Pichia pastoris X-33 as the host cells, proceed the fuse-expression of micromolecule antibody ScFv-Fc.Base on the former experiments of laboratory, we get VH and Vκgene of human antibody by PCR, through SOEing PCR we gain ScFv, and linkage it with the vector pPICZα-Fc which is constructed in our laboratory had a Fc in it. By analysing and indentifying the anyibody expressed, assesss the valence of ScFv-Fc and the function of the Pichia pastoris expression system in the micromolecule antibody cloning, filtrating, expression, and fermentation, offer the theoretical and experimental foundation of the exploitating and development of remedial monoclone antibody.Experiment contents:1. Use PCR to clone human antibody variable region of theheavy chain and the light chain (VH,Vκ)Extracting the plasmid which contain the human antibody gene from the affordable strain. Design primers acoording to the known-human antibody sequence,use PCR to gain human antibody variable region of the heavy chain and the light chain (VH,Vκ). By agarose electrophoresis,we can validate the size of the gene fragment. 2. Construction of ScFv antibody expression systemReclaim the product of PCR (VH and Vκ) by using gel reclaiming kit. Put the VH, Vκand Linker (get from our lab) constitute into ScFv by SOEing PCR. Amplify the ScFv we get by PCR, and reclaim regularly. Use the Xho I+Apa I to assimilate the production, connect it regularly with pPICZα-Fc expression vector. Select 20 monoclone colony from Zeocin+ plate randomly, extract the plasmid, use the Xho I+Apa I assimilate the pPICZα-Fc/ScFv to identify。3. Expression of ScFv-Fc antibdy in Pichia pastorisWe amplify the monoclone strain, then extract pPICZα-Fc/scFv plasmids by using the kit. Plasmids 15 g, PmeI assimilate,extract by hydroxybenzene and chloroform. Deposite by ethaol and dissolve by 15 l double-disdilled water,then electrotransformate it into pichia pastoris. We use Zeocin+ YPD plate to sieve masculine clone, methaol derivate to express, build a ScFv-Fc pichia pastoris fuse expression system. The expression continued three round, we use the anti-rabies antibody ELISA test kit from Lanzhou Biological Product Institute to test the expression supernatant for firstly protein activity screening. The supernatant from the strain in which results displayed positive, we got it, progress PCR, SDS-PAGE(10% separate gel, 5% concentrate gel) and Western-blot to test the expression.4. Purification and identification of ScFv-FcPut the yeast strain RSC10 which expresses the anti-rabies antibody into 1L BMGY to amplify, 30℃shake cuture 24h, when OD600 reach 2.0～6.0, collect the cells. Use BMMY which is the same volume to make the cells resuspension. Induce to express at 30℃. During the process, add methanol to make it at a 0.5% final concentration. Centrifuge the cells at the fifth day. We use (NH4)2SO4 to concentrate the supernatant, then purify by Protein A Sepharose, we use SDS-PAGE and Western-blot to analyze the product.Results:1. Use PCR to clone human antibody variable region of theheavy chain and the light chain (VH, Vκ)We check the product by agarose electrophoresis and we get two bands at about 370bp (VH) and 325bp (Vκ) as same as we forcast, firstly identification is conreect.2. Construction of ScFv antibody expression system Put the VH, Vκand Linker (get from our lab) constitute into ScFv by SOEing PCR. We check the product by agarose electrophoresis and we get a band at about 750bp as same as we forcast, firstly identification is conreect. We extract the plasmids, use the Xho I+Apa I to assimilate pPICZα-Fc/scFv for identification,we get fragment about 750bp and 1500bp, demonstrate that ScFv fragment insert successfully.3.Expression of ScFv-Fc antibdy in Pichia pastorisWe use the anti-rabies antibody ELISA test kit from Lanzhou Biological Product Institute to test the expression supernatant for firstly protein activity screening. We got 12 positive anti-rabies antibody strain, use boil-freeze-boil method to extract the genome DNA of positive yeast strain that test by ELISA, use the VH,Vκ primers to amplify them by PCR, ligate the product into pMD-T vector, then we got a correct result by DNA sequencing. After SDS-PAGE and Western-blot, wo got a dyeing band at about 52kD.Ensure the secretive expression of ScFv-Fc micromolecule antibody which has the activity of combining with rabies virus.4. Purification and identification of ScFv-FcPut the yeast strain RSC10 which expresses the anti-rabies antibody into 1L shake flask to amplify, 0.5% methanol to induce, we use (NH4)2SO4 to concentrate the supernatant, then purify by Protein A Sepharose, we use SDS-PAGE and Western-blot to analyze the product. Western-blot results show the band at about 54kD(relative molecular weight), that demonstrate the expression of ScFv-Fc. |
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