The Secretory Eukaryotic Expression Of Extracellular Fragment Of Hepatoma Associated Antigen HAb18G

HAb18G, a hepatoma associated antigen, is a heavy glycosylated membrane protein. Using the monoclonal antibody HAbl8 which have been prepared by our lab we have cloned its cDNA sequence from the cDNA library of hepatoma. Its open reading frame sequence is identical with CD147 molecule's, and it is the same protein as Extrcellular Matrix metalloproteinase Inducer(EMMPRIN), Basigin, Nureothelin but expressed in different tissues or cells. We have constructed HAblSG eukaryotic expression vector and have expressed it in COS-7 and CHO cells successfully, which make a basis of the further research of this protein. However, besides maintaining the protein' s bioactivity, it is necessary to obtain a large amount of the antigen for research. It is very difficult to obtain a large quantity of the membrane protein because of complication of its purified process. Moreover,lysis solution and other chemical or physical factors may lead to some change of protein's bioactivity or structure, which make it uneasy for research. Secreted protein can be harvested from cells culture medium directly and repeatly. Using molecular biology technology to reconstruct the gene of membrane protein, we can make it be a secreted protein which can be got simply. The aim of this experiment is to construct a eukaryotic secretory expression vector of HAb18G gene extracellular fragment and expressed in COS-7 cells. Try to find a more simple way to gain this protein and lay the foundation of its functional research.By deleting the sequence encoding transmembrane domain and cytoplasmic domain , HAb18G extracellular fregment was subcloned into an eukaryotic expression vector pcDNAS. The recombinant plasmid then was transfected into COS-7 cells by mixing with lipofectamine 2000 reagent . Then the supernatant was harvested and then purfied by affinity chromatography, and detected by SDS-PAGE electrophoresis, DOT-ELISA, ELISA and Western blot. RT-PCR was used to identify the expression of transfected COS-7 cells. The results show that the eukaryotic secreted expression vector pcDNAS containing gene encoding extracellular fragment of hepatoma associated antigen HAblSG has been constructed successfully and its open reading frame was confirmed being inserted correctly by nucleotide sequencing and restriction endonuclease digestion. RT-PCR confirmed that HAblSG had been expressed in COS-7 Cells. DOT-ELISA and ELISA had detected the expression of the protein a, SDS-PAGE electrophor- esis has confirmedhad detected the expression of the protein a, SDS-PAGE electrophor-esis has confirmed that its molecular weight is 45 KDa. But the result of western blot is negative. Conclusion: By deleting the sequence encoding the cytoplasma domain and transmembrane domain and expressing in COS-7 cells, we have obtained a secretory type protein of HAblSG, which have the correspondence antigen activity . These results can be used for studying its biological function and obtaining a large amount of expressed protein by a simple way.