The Construction And Expression Of Engineering Antibody Chimeric IgG And Chimeric Fab Against Human Hepatoma

To high efficiently express the extracellular domain of hepatoma associated antigen HAblSG in E.coli and check the immunologic characteristics of expressed protein so as to assure whether it can be used as antigens in identifying and selecting specific engineering antibody. Then, to clone Fd and light chain genes and VH and VL genes of monoclonal antibody HAb 18 against human hepatoma and verify their accuracy and liability. And then to construct a eukaryotic expression vector pDHL/chHAbl8 of chimeric IgG antibody by use of cloned VH and VL genes and to transfect it to meyloma cell line SP2/0 to get stable expression of chHAb18. To insert VH and VL genes orderly into an universal expression vector of human-mouse chimeric Fab antibody to construct a secretory expression vector pComb3/cFab. Then, to transform it into E.coli to express cFab antibody by IPTG induction. At the same time, two non-fusion prokaryotic expression vector pET32a/cFd and pET32a/cL will be constructed to express chimeric Fd and chimeric light chain of MAb HAb18 by induction in E.coli. Then, to refold chimeric Fd and chimeric light chain together into cFab and perform the identification of renatured products. Finally, to confirm the relative antigen binding ability of three kinds of engineering antibody and make a affinity comparison between them and parental mouse antibody.Methods: All the studies can be divided into six parts according to experiments' aims. The protocols were briefly described as the following:1.Prokaryotic expression and immunological Analysis of the Extracellular domain of Hepatoma associated antigen HAb18G The gene of extracellular domain of hepatoma associated antigen HAblSG was amplified by PCR with pBluescript KS(+)/HAb18G as the template. Then, the cloned gene was inserted into a prokaryotic expression vector pGEX-4T-3. The positive E.coli clone containing recombinant plasmid pGEX-4T-3/HAb18GE was selected and identified by restriction endonuclease digestion and sequencing analysis and induced to express fusion proteins by IPTG. Whether high efficient expression was fulfilled can be checked by SDS-PAGE electrophoresis and Western Blot analysis. After the purification and refolding, the immunological characteristics of expressed fusion protein can be analyzed through ELISA under the condition of denaturation and renaturation2.Cloning and identification of the heavy and light chain genes of MAb HAb18 against human hepatoma Firstly, the total RNA was extracted from hybridoma cell, and the Fd and light chain genes and VH and VL genes of MAb HAb18 were amplified by RT-PCR. After the cloned genes were inserted into cloning vector pMD18T, DNA sequencing was done. Then, all obtained DNA sequences were analyzed by corresponding software. Secondly, the light chain and Fd genes were sequentialy cloned into pComb3 to construct expression vector pComb3/Fd-gIII-L which could be used to display Fab on the surface of phages. Moreover, this vector was transformed into E.coli XL 1-blue and rescuing was done with helper phage M13K07. Phages were prepared by centrifugation and their antigen binding specificity was detected by indirect ELISA. Finally, the vector pComb3/Fd-gIII-L was cut by restriction endonuclease enzyme to remove gIII gene and then was ligated to a new secretory expression vector pComb3/Fab. After recombinant vector was transformed into E.coli, inductive expression by IPTG was conducted. The expression products were detected by ELISA and immunofluorescense staining to confirm its specificity.3.Construction and expression of engineering chimeric IgG antibody against human hepatoma The VH and VL genes of MAb HAb18 were PCR amplified by specific primers containing restriction endonuclease sites and intron splicingsite with cloning vector pMD18-T/VH and pMD18-T/VL as templates. After cloned genes were inserted into eukaryotic expressing vector pDHL, positive recombinant vector pDHL/chHAb18 was checked by restriction endonuclease enzyme digestion and sequencing. Then, vector pDHL/chHAb18 was transfecte...