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The Eukaryotic Expression of HHV-8 K1 and Its Effect on the Proliferation of EC Cells and PC-3 Cells

Author: ZuoZhiChao
Tutor: HuaLiXin
School: Nanjing Medical University
Course: Urology
Keywords: HHV-8 K1 oncogenic gene Eukaryotic expression Prostate Cancer
CLC: R737.25
Type: Master's thesis
Year: 2011
Downloads: 3
Quote: 0
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Abstract


Objective: To construct containing human herpesvirus 8 (HHV-8) K1 oncogenic gene eukaryotic expression plasmid into human vascular endothelial cells (EC) and prostate cancer (PC-3) for expression , and to explore K1 protein on PC-3 cells and EC proliferation . Methods: K1 gene registered in GenBank nucleic acid sequence to design PCR primers at the 5 'end and 3' ends of the introduction of XhoI and XbaI restriction sites and downstream sequences into Flag -tagged protein expression after- K1 testing. K1 gene containing an HHV-8 DNA as template K1 gene was amplified by double digestion after it was cloned into the eukaryotic expression vector pCI-neo in . Recombinant plasmid was identified by nucleic acid sequencing EC cells were transiently transfected and PC-3 cells 48 h after transfection, total RNA was extracted and total protein , respectively, by RT-PCR and Western blot K1 gene transcription and expression . Finally, we use MTT and flow cytometry (FCM) detection K1 protein PC-3 cells and EC proliferation . Results : We successfully constructed a recombinant plasmid containing K1 pCI-K1, nucleic acid sequence analysis showed that the cloned K1 gene is 928 bp, registered in GenBank K1 gene 100% homologous to the introduction of the Flag sequence is correct. The pCI-K1 transfected EC cells and PC-3 cells can be detected in K1 K1 mRNA and protein . MTT and flow cytometry K1 protein found to promote PC-3 cells and EC cells. CONCLUSION : The recombinant plasmid pCI-K1 cells were successfully constructed and the EC and PC-3 cells expressed correctly . K1 protein to promote PC-3 cells and EC cells.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Male genitalia tumors > Prostate cancer
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