The Eukaryotic Expression Of HHV-8 K1 And Its Effect On The Proliferation Of EC Cells And PC-3 Cells

Objective: To investigate the expression of the recombinant plasmid of HHV-8 K1 both in the EC cells and PC-3 cells, and to evaluate the effect on the proliferation of PC-3 cells and EC cells by K1 protein. Methods:A pair of PCR primers for K1 gene including XhoI and XbaI restriction enzyme cut sites was designed according to the sequence registered in GenBank. Then the genome of HHV-8 was taken as template and K1 gene was amplified applying PCR; Subsequently, amplified gene fragments were digested with the two enzymes mentioned above, A Flag sequence was introduced to facilitate the detection of K1 protein After identification with nucleotide sequences analysis, and then cloned into pCI-neo vector to generate recombinant eukaryotic expression plasmid designated as pCI-K1, recombinant pCI-K1 was transient transfected into EC cells and PC-3 cells. The expression of pCI-K1 mRNA and protein in the two cell lines were detected by RT-PCR and Western blot. The MTT and FCM methods were used to detect the effect on the proliferation of PC-3 cells and EC cells by K1 protein. Results:Nucleotide sequences analysis indicated that the isolated and cloned pCI-K1 sequence length was 928 bp. The isolated K1 sequence was 100% homology with K1 gene registered in GenBank. The specific bands were both detected at the expectant place by RT-PCR and Western blot. The results of MTT and FCM disclosed that the K1 protein could promote the proliferation of PC-3 cells(P<0.05)and EC cells(P<0.05). Conclusion: K1 gene could be correctly expressed in EC cells and PC-3 cells, and the K1 protein could promote the proliferation of EC cells and PC-3 cells.