[Development of a system for quick screening of efficient HBx-siRNA].
To develop a system for quick screening of efficient siRNA targeted HBx mRNA.



Using recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis.



(1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388.



Combination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx-  siRNA and its matching promoter.
Ni
Qin
Q
Institute of Infectious Diseases, The First Affiliated Hospital, Medical College, Zhejiang University, Hangzhou 310003, China.
Liu
Ke-zhou
KZ
Yang
Zheng-gang
ZG
Yao
Hang-ping
HP
Ai
De-hua
DH
Li
Min-wei
MW
Chen
Zhi
Z
D001483:Base Sequence; D002478:Cells, Cultured; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D034741:RNA, Small Interfering / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D049452:Green Fluorescent Proteins