The Exploration Of Copine-8 Function And The Preparation Of Its Polyclonal Antibody

Copine-8 is one of the members of copines protein family.This protein family is an identified class of calcium-dependent,phospholipid binding proteins which expresses in many kinds of species including animals,plants and so on All the members of copines family share some sort of similar structures:two similar C2 domains and an A domain.The biological function of copine-8 is still unclear.So in order to know the biological function of copine-8,this study aims to explore the sub localization of copine-8 and its function in tumor cells preliminarily and preparing the anti-copine-8 antibody.Hela cells were grown in complete DMEM medium and were then harvested after trypsin treatment.Total RNA was extracted from Hela cells with Trizol reagent.Copine-8-encoding DNA fragment(CPNE8)was amplified by RT-PCR.CPNE8 DNA fragment was cloned into pEGFP-Nl and pGEX-4T-1 respectively by using conventional ligation method(restriction digestion,ligation were required).And the subsequent ampicillin resistant transformants were screened by PCR amplification to identify the plasmids containing the inserts.Agarose gel electrophoresis showed a high yield of total RNA,as well as a distinct band of RT-PCR product that identified as CPNE8 by DNA sequencing.The recombinant vectors pEGFP-N1/CPNE8 and pGEX-4T-1/CPNE8 have been constructed.And the both recombinant vectors had been identified through the sequencing method.H1299 cells had been transfected with the eukaryotic expression vectors pEGFP-N1/CPNE8.According the requirement,these H1299 cells were cultivated again and then stained.And then using statistics method the numbers of cells between experimental groups and control groups(H1299 cells transfected with pEGFP-N1)were commpared.At the same time,the numbers of migration of H1299 cells between experimental groups and control groups were compared by transwell method.And then 293T cells had been transfected with the eukaryotic expression vectors pEGFP-N 1/CPNE8.The subcellular location of the copine-8 protein overexpressed in 293T cells were observed by confocal laser scanning microscopy.And then the eukaryotic expression vector pEGFP-N 1/CPNE8 had been transfected into H1299 cells.The subcellular location of the copine-8 protein overexpressed in H1299 cells also had been observed.The prokaryotic expression vectors pGEX-4T-1/CPNE8 were transformed into competences of E.coli BL21(DE3)and E.coli Rosetta(DE3)and then the expression optimal conditions had been identified.GST-copine-8 fusion protein had been expressed through IPTG induction and this fusion protein had been purified.The purified GST-copine-8 fusion protein was used to immunize the New Zealand white rabbits.And then we got the titer of anti-copine-8 antibody.Using the cell colony formation assay showed that the growth of H1299 cell with copine-8 overexpression increased markedly compared with the control group.The result showed that transient overexpression of pEGFP-N1/CPNE8 in H1299 could promote the ability of proliferation of lung adenocarcinoma cells in vitro.Tranwell assay showed that the number of migrated H1299 cells with copine-8 overexpression reduced markedly compared with the control group.The result showed that transient overexpression of copine-8 in H1299 could suppress the ability of invasion of lung adenocarcinoma cells in vitro.By observing subcellular location of the copine-8 we found that copine-8 mainly located in the plasma membrane and a few in the cytoplasma of 293T cells while most of copine-8 located in the plasma membrane of H1299 cells.The ELISA results revealed that the titer of anti-copine-8 antibody raised from rabbit reached about 1:10000 after the 4th immunization Western blotting showed that this antibody could specifically recognize the copine-8 protein.At the same time the anti-copine-8 antibody could be used in immunohistochemical technology(IHC)for discovering the distribution of copine-8 in different issues from human.