Expression Of Bovine Scrapie Prion Protein Gene PrP102-242 And PrP1-264 In COS-7 Cells

【Objective】PrPSc (Scrapie Prion protein) is thought to be a pathogenic factor of transmissible spongiform encephalopathy(TSE) including Bovine Spongiform Encephalopathy(BSE) which is transformed by a normal PrPC(cellular prion protein), PrP27-30 is the infectious core of PrPSc, PrPC is encoding a normal endogenous gene Prnp. Investigation into the structure and function of PrPC is useful to elucidate the pathogenic mechanism of PrPSc. Although PrPC exists in brain tissue, the difficutes of extract limited the prograss of reseach. Obtaining onformation of PrP using the mammalian cell expression system will accelerate reseach of PrPC. The purpose of this study is to achieve recombinant protein of PrP102-241 (mature PrP, encoded PrP27-30 ) and PrP1-264 (consised of the mature PrP, N terminal signal peptide and C terminal GPI anchored form) expressed in COS-7 cells.【Method】The gene of PrP102-242 and PrP1-264 are obtained from the recombinant vectors of pMD18-T-PrP102-242 and pMD18-T-PrP1-264 by PCR,and digestion of production of PrP102-242, PrP1-264, expression vector pCI-neo was digested by same restriction endonuclease and ligated with PrP102-242, PrP1-264 to construct two recombinant expression vectors, respectively. The two recombinant vectors were identified by PCR, enzyme digestion , and squence analysis PCR product of two recombinant vectors. two recombinant plasmids were transferred into COS-7 cells by liposome and stable expression COS-7 cell lines were obtained by screening with 100ug/mL, 200ug/mL, 300ug/mL and 400ug/mL G418. The transcriptional and expressional level of two PrP102-242, PrP1-264 detected by RT-PCR ,indirect immunofluorescence, indirect ELISA and Western-blot, with anti-PrP monoclonal antibody (7B6/D2)as specific probes.【Results】RCR, double enzyme disition and squence anylysis indicated that the resulting plamids of pCIneo-PrP102-242 and pCIneo-PrP1-264 were correctly inserted pCI-neo vector. Two resistance cell stains, named 7B2 and 30C1, respectively, were obtained using 4 different concentrations of G418 and subcloning method. RT-PCR analysis indicated that the recombinant plasmid carried exogenous genes have been integrated into COS-7 cell genome and can be transcribed in cells. Indirect immunofluorescence testing suggested that target genes can be expressed in the COS-7 cells . The results of indirect ELISA showed that OD value of lysate of 2 times diluted of 7B2 and 30C1 is 1.095 and 1.259, respectively. When the concetration of G418 range from 100ug/mL to 400ug/mL, expression level of 7B2 and 30C1 wre positively correlated with the concentration of G418.SDS-PAGE and Western-blot analysis indicated that size of 7B2 and 30C1 are 18.0kD and 35.0kD in COS-7 cells,which is in consistent with the expected size of target protein, and lysate of 7B2 and 30C1 can react with PrP monoclonal antibody(7B6/D2) specificly.【Conclusion】In summary, we constructed two mammalian expression vector pCI-PrP102-242 and pCI-PrP1-264 harboring different length PrP gene. Obtaining two postive cell lines of 7B2 and 30C1 whose ELISA value is more high than other selected cell lines by screening of different concentration of G418. The cell line of 7B2 and 30C1 could express the PrP102-242 and PrP1-264, respectively. The indirect immunofluorescence, indirect ELISA and Western-blot analysis showed that 7B2, and 30C1 could react with PrP monoclonal antibody (7B6/D2) specificly.