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1、VIROLOGICA SINICA, October 2007, 22 (5):366-373 CLC number: Q78 Document code: A Article ID: 1674-0769(2007)05-0366-08 Received: 2007-04-05, Accepted: 2007-07-09 * Foundation items: National Natural Science Foundation of China ( No.30571643, No.30672380) and National Key Basic Research Program of Ch
2、ina ( No. 2005CB2401, 2007CB512904) # These authors contributed equally. * Corresponding author. Tel: +86-27-83663181, Fax: +86-27-83662391, E-mail: Construction of shRNA of Fulminant Hepatitis Related Gene mfgl2 and Investigation of Its Biological Effects in vitro* Dong XI#, Zhi-Mo WANG#, Sui GAO,
3、 Chuan-Long ZHU, Jian-Wen GUO, Xiao-Ping LUO and Qin Ning* (Laboratory of Infectious Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China ) Abstract: This study was designed to explore the RNA interference technique in inhibition o
4、f the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA, complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA
5、(shRNA) which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDN
6、A3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA o
7、n mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro. Key words: Fulminant hepatitis; fgl2 prothrombinase; RNA
8、interference; shRNA Fibrinogen like protein 2 (fgl2) which encodes a serine protease is capable of directly cleaving pro- thrombin to thrombin to trigger the process of coagulation. In mice with fulminant viral hepatitis induced by murine hepatitis virus strain 3 (MHV-3) and patients with severe acu
9、te or chronic hepatitis B, it has been shown that the high expression of fgl2 results in intravascular fibrin deposition within the liver, culminating in widespread hepatocyte necrosis, and is highly correlated with disease severity (3, 10, 12). Fgl2 plays an important role in the development of MHV
10、-3 induced fulminant hepatitis and severe XI et al. Construction of mfgl2 shRNA plasmid and investigation of its biological effects 367 acute on chronic hepatitis B in patients. The pharmacological blockage of fgl2 may offer an important new therapeutic approach in hepatitis virus induced disease. R
11、NA interference has proven to be an extremely potent and versatile tool to specifically reduce expression of targeted genes. Research reports demon- strate the potential for use of small interfering RNAs (siRNAs) as therapeutic agents, especially in the areas of cancer and viral infection. siRNAs ca
12、n be surprisingly effecient, for example, transfections done using subnanomolar concentrations of RNA some- times achieve 90% reduction in mRNA levels (7). In this paper, a expression plasmid containing short hairpin RNA (shRNA) of mouse fgl2 (mfgl2) was constructed and its interfering effect was in
13、vestigated in vitro. MATERIALS AND METHODS Construction of p-mfgl2shRNA plasmid Mouse U6 promoter (U6P) was amplified from genomic DNA extracted from mouse liver. The upstream primer (primer 1) was 5-GTAGGATCCATC CGACGCCGCCATCTCTA-3 and the downstream primer (primer 2) was 5-GGCAGCCAAGCTTCACA AACAAG
14、GCTTTTCTCCAA-3. The boldface, under- lined sequences correspond to the restriction enzyme sites for BamH I and Hind III, respectively. The amp- lified fragment was cloned to T-vector pMD-18 (Invitr- ogen Life Technologies, Carlsbad, USA) to construct pMD-18-U6. A 19 bp oligonucleotide (5-GCAGTG GACA
15、GTCTGAAGA-3, S1) in the exon1 of mfgl2 and its inverted repeat (5-TCTTCAGACTGTCCA CTGC-3, S2) formed the double strand of the constructed shRNA. The constructed shRNA was 52 bp and its sequence was 5-GCAGTGGACAGTCTG AAGATTCAAGAGATCTTCAGAGTGTCCACTGCTTTTT-3 (S1+TTCAAGAGA+S2+TTTTT), which was produced
16、by 3 rounds of PCR with following primers: primer 3: 5-CTTCAGACTGTCCACTGCAAACAA GGCTTTTCTCC-3, primer 4: 5-AGTCTGAAGATCTCTTGAATCTTCA GACTGTCCACTGC-3, primer 5: 5-AAGCTTAAAAAGCAGTGGACAGTC TGAAGATCTCTTGAAT-3 (Fig.1). By using the transfect vector pMD-18, U6P and shRNA template were subcloned into pMSCVneo vector (Invitrogen Life Technologies) at the BamH I and Hind III restriction sites to construct the plasmid
