ABSTRACT
Objective To study the expression and biological functions of RN181 in SMMC7721 cells,the retroviral vector was constructed.Methods Gene cloning techniques were used to construct pRetrox-TRE3G/RN1S1 recombinant vector.The regulating plasmid pRetroX-TRE3G/RN181 or the response plasmid of pRetroX-Tet3G were respectively cotansfected into GP2-293 cells with Envelope Vector plasmid to package retrovirus after routine identification.Both viruses co-infected target cells SMCC7721,and then were selected by G418 to obtain stable cell lines.The stable cell lines were induced by doxycycline (DOX),and then verified by RT-PCR and Western blotting.CCK-8 was used to evaluate the effect of RN181 on growth of SMMC7721 cells.Results We constructed the recombinant plasmid.Stable recombinant plasmid were verified by screening.And there were significant differences of RN181 between the induced and uninduced cell lines through RT-PCR and Western blot.Conclusions We have successfully constructed the inducible stable RN181 expression SMCC7721 cell,which can be used as an effective cell model to study the biological functions of RN181.We found RN181 could suppress the proliferation and invasion in SMMC7721 cells in vitro.
ABSTRACT
Objective To prepare the PSMA7 hybridoma cell lines using classical hybridoma technique and to perform purification and elementary identification of the monoclonal antibodies(McAbs)against PSMA7 antigen for further study.Methods BALB/c mice were immunized with purified recombinant PSMA7 protein.Once the antibody titer of blood taken from mouse tail reached 1∶6 400,a cell fusion was performed between mouse splenic cells and myeloma cells(Sp2/0),and then the hybridoma cell lines secreting monoclonal antibodies against PSMA7 antigen were screened by indirect enzyme linked immunosorbent assay(ELISA).The immunoglobulin subtypes and titer of the monoclonal antibodies against PSMA7 antigen were identified and measured by Western blot analysis and enzyme linked immunosorbent assay,respectively.Results After cell fusion and subcloning,two hybridoma cell lines that stably secreted monoclonal antibodies against PSMA7 antigen were successfully obtained:B013 and B001,which belong to the subtypes of IgG1.The antibody titers in the hybridoma culture supernatant were 1∶128 and 1∶256,and those in the ascites fluid were 1∶25 600 and 1∶32 000,respectively.Western blot analysis and immunohistochemistry showed that the two antibodies can specifically bind with PSMA7 antigen derived from human eucaryotic cells or tissue.Conclusions Two monoclonal antibodies against PSMA7 antigen with high titers and specificity have been successfully prepared.These antibodies can be used for the identification of PSMA7 protein,and may be a useful tool for studying the biological properties of PSMA7 protein.