[Establish a screening system for selection of mRNA target sites for HBsAg to construct siRNA with shRNA].
OBJECTIVE
To find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors.


METHODS
Four shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR.


RESULTS
Four shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%.


CONCLUSIONS
The results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells
Yang
Zheng-Gang
ZG
Institute of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
Chen
Zhi
Z
Xu
Ning
N
Ni
Qin
Q
Pan
Xiu-Cheng
XC
Jin
Han-Ying
HY
Li
Min-Wei
MW
D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D034743:RNA-Induced Silencing Complex / Q000235:genetics
D006514:Hepatitis B Surface Antigens; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex