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The Study of Inhibtion Effect by shRNA Expression Vector on Herpes Simplex Virus Type 2 UL54

Author: FanJun
Tutor: LvYanCheng
School: Zunyi Medical College,
Course: Biochemistry and Molecular Biology
Keywords: RNA interference small hairpin RNA Herpes simplex virus type 2 UL54 gene
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 1
Quote: 0
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Abstract


Objective:The expression vector of small hairpin RNA (shRNA) target herpes simplex virus type 2 (HSV-2) UL54 gene were constructed and transfected Human Embryonic Kidney 293 cells (HEK293). Influence of UL54 gene expression and replication of HSV-2 in HEK293 cell by expression vector expressing shRNA was observed in cell.Methods:Five interference target sites of HSV-2 UL54 gene were selected. expression vector expressing shRNA target UL54 gene named shRNA1, shRNA2, shRNA3, shRNA4 and shRNA5 respectively. The negative control expression vector expressing shRNA NC was designed also. The expression vector transfected into HEK293 cell with liposomes. HEK293 cell were infected with HSV-2 after expression vector transfectied 48 hours. The transcription levels of HSV-2 UL54 were determined using Real time PCR. The viral titer were estimated using end point assay.Results:(1) The results of DNA sequencing and restrictive enzyme digestion confirmed that recombinant plasmid of shRNA, shRNA2, shRNA3, shRNA4 and shRNA5 were successfully constructed. (2)The result of Real-Time PCR showed the expression level of UL54 mRNA is depressed by shRNA1, shRNA4 and shRNA5 recombinant plasmid in different extently(P<0.05). (3) The result of end point assay showed the virus titers of the supernatant was reduced by shRNA1, shRNA4 and shRNA5 recombinant plasmid in different extently(P<0.05).Conclusion:The expression vector of small hairpin RNA target herpes simplex virus type 2 UL54 gene were were successfully constructed. The shRNA11, shRNA4 and shRNA5 recombinant plasmids could interfer the expression of UL54 gene and inhibit the replication of HSV-2 genome in HEK293 cells. which will provide an experimental foundation for the further investigation of anti-HSV-2 gene therapy by RNA interference.

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