Abstract: In order to further study the physiological function of bovine prion protein and anti-mad cow disease cloned transgenic cattle basis form cellular level, we constructed a bovine prion protein gene (PRNP) eukaryotic expression vector. The PRNP gene was cloned by splicing overlap extension PCR (SQE-PCR) method, and inserted into the report with DsRED2 eukaryotic vector pDsRED2-N1.This recombinant plasmid pDsRED2-N1 was identified by digestion of endonuclease, PCR and sequencing. Then, it was transfected into bovine BMSC cell line mediated by Lipofectamine^TM2000. The transfected cells was detected by fluorescence microscopy and drug screening by 800 μg/mL G418. Electrophoresis analysis showed that the sequences were amplified and the sizes of products were accord with expectation. The recombinant plasmid was expressed in BMSC. The positive single stable transfection cell clones were also got by drug screening. The PRNP gene had been cloned by splicing overlap extension PCR method, and the recombinant eukaryotic vector stable expressed in BMSC successfully.

Key words: mad cow disease; PRNP gene; splicing overlap extension PCR; BMSC