Eukaryotic Expression Of CSFV P80 Gene And CPE Of P80 Protein On PK-15 Cells

With the method of reverse transcription-polymerase chain reaction (PT-PCR) and nest PCR(nPCR), P80 gene of CSFV is cloned from the cells infected by the strain Shimen of csfv, and connected with eukaryotic expression vector pEGFP-C1. Then recombined eukaryotic expression plasmid pEGFP-P80 of P80 gene is achieved successfully. It builds the basis for expressing and gaining protein p80 in mammiferous cells. At last, the recombined plasmid pEGFP-P80 is tansfected into the PK-15 cells and expressed for the study of the toxic effect of protein p80 on the PK-15 cells. All of these give the deeper discuss about the relationship between p80 protein and appearance of CPE of cells from the kidney of pigs. 1. According to the whole nucleotide sequences of the strain Alfort and Brescia published, the two pairs of primers, P1/P2 and B1/B2, are designed. The two sites for the recognition of the enzymes Xho I and Apa I are added to the end 5'of B1 and B2, respectively. P80 gene, the nucleotide sequence of about 2.0kb, is gotten with the methods of RT-PCR and nPCR by One-Step RNA Extract kit. The PCR product above is separated, purified and cloned onto the vector pMD 18-T. Then it is sequenced, PCR and digested by the restriction enzymes Bamh I /Hind â…¢. The results show that it is the recombined plasmid pMD-P80 of P80 gene. Then pMD-P80 is digested by the enzymes Xho I and Apa I, separated and linked to the linear plasmid pEGPF-C1 after digested by Xho I and Apa I. So the recombined expression plasmid pGFP-P80 is achieved. Then it is sequenced, PCR and digested by the restriction enzymes Xho I and Apa I. The results give that pGFP-P80 is achieved successfully and the insert sites, direction and reading frame are all right. It builds the basis for expressing, purifying, gaining p80 protein from mammiferous cells. 2. By the electropositive Liposome 2000, in the 6-plate for cell culture, pEGFP-P80 above is transfected into the PK-15 cells from the kidney of pigs and expressed. During the 6th h after the transfection, the green fluorescence given off by the fuse proteins of pEGFP-P80 can be observed in the transfected cells under the fluorescence microscope; During the 24th h after the transfection, by immunohistochemical method SABC, the interested protein p80 in the transfected PK-15 cells can be found effectively. These show that recombined eukaryotic expression plasmid pEGFP-P80 can express p80 protein with the fine immune activity in PK-15 cells. Repeat the transfection: in the 96-plate, the plasmid pEGFP-P80 and pEGFP-C1 are respectively transfected into the two groups of PK-15 cells. The fist is the testing group, and the second is the control. Respectively, during the 24 h and 48 h after the transfection, the two groups of cells are stained by MTT and mensurated OD when the absorption wave length is 630nm. Both activities of the two groups of the cells are analysized. With the comparition of the two groups, the conclusion can be drawn that the p80 protein expressed by the plasmid pEGFP-P80 cannot cause the detectable depression of activity of PK-15 cells.