Plasmid containing HCV genome, cell, system and method for screening drug
A technology of hepatitis C virus and whole genome, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of reducing stability, limiting wide application, and inability to obtain stability Solve problems such as cell lines that secrete HCV locally, and achieve the effects of improved stability, convenient storage, easy operation and repeatability
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Embodiment 1
[0040] Construction of embodiment 1 plasmid pEGFP-JFH1-Ribozyme
[0041] Due to the large size of the plasmid pJFH1, it is difficult to directly introduce the ribozyme sequence by mutation PCR, so the design is as follows: first, a part of the sequence in pJFH1 is excised by enzyme digestion, and then the ribozyme sequence is introduced to obtain the early stage plasmid pJFH1-dNheI+Ribozyme (step 1); then reinsert the excised HCV sequence through two connections to obtain the prokaryotic vector pUC19-JFH1-Ribozyme (steps 2 and 3); finally, digest the prokaryotic expression vector obtained in the previous step, reclaim the target fragment, and connect into the eukaryotic expression vector pEGFP-N1 to obtain the target plasmid pEGFP-JFH1-Ribozyme (step 4).
[0042] The specific operation is as follows:
[0043] 1. Construction of the early stage plasmid pJFH1-dNheI+Ribozyme
[0044] The original plasmid pJFH1 was single-digested with NheI, and the 3001bp fragment was recovered...
Embodiment 2
[0051] Example 2, construction of control plasmid pEGFP-JFH1-Ribozyme / GND
[0052] On the basis of pEGFP-JFH1-Ribozyme, the 8618th base in the HCV genome sequence was mutated from G to A, so that the encoded amino acid was mutated from D (GAT) to N (AAT), which made it The encoded product loses RNA polymerase activity. The control plasmid pEGFP-JFH1-Ribozyme / GND (such as image 3 ). For specific operation methods, see "(One-step site-directed mutagenesis using DREAM design and homologous recombination", China Biotechnology Journal, 2008, 28(11): 77-81.
Embodiment 3
[0053] Embodiment 3, transfection
[0054] The plasmid pEGFP-JFH1-Ribozyme with self-cutting function prepared in Example 1 and the control plasmid pEGFP-JFH1-Ribozyme / GND prepared in Example 2 were transfected into HepG2 cells and / or Huh7 cells respectively, according to Lipofectamine2000 (Invitrogen Corporation ) instruction manual. After 24 hours of transfection, G418 solution (selection solution containing neomycin, purchased from Sigma) was added to a final concentration of 0.7 g / L (the optimal concentration determined for the preliminary experiment). Since the above plasmid has a neomycin selection marker, only the cells integrated with the plasmid can survive in the medium containing G418, so as to obtain monoclonal cells, and select the target by RT-PCR, immunohistochemistry, immunofluorescence and other methods. Monoclonal cell lines HepG2 / GDD and HepG2 / GND.
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