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Cloning and Expression of the Fusion Gene Encoding Hyper-IL-6, and Generation of the Recombinant Adenovirus AdHIL-6

Author: ZuoChunHua
Tutor: QinBo
School: Chongqing Medical University
Course: Internal Medicine
Keywords: Hyper-IL-6 adenovirus fusion gene gene therapy
CLC: R346
Type: Master's thesis
Year: 2005
Downloads: 28
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Abstract


PARTⅠ CLONING AND SEQUENCE ANALYSIS OF HUMAN SOLUBLE INTERLEUKIN-6 RECEPTOR Objective To clone the human soluble interleukin-6 receptor gene from the human hepatoma cell lines HepG2 stimulated by dexamethasone and make it possible to construct the fusion gene Hyper-IL-6. Methods Total RNA was isolated from the stimulated HepG2 cells. The sIL-6R gene was amplified RT-PCR using the primers based on the published sequence of sIL-6R. The PCR product was purified by gel extraction and cloned into pIRES2-EGFP. The recombinant plasmid was screened by restriction analysis and sequencing. Results Sequencing analysis revealed that the amino acid sequence of sIL-6R gene is identical to the published sequence in Genebank although there are two variations in nucleotide sequence. Conclusion We have successfully cloned the human sIL-6R gene. sIL-6R; gene;gene cloning Hyper-IL-6; green fluorescence protein; eukaryotic cell; expressionPART Ⅲ CONSTRUCTION OF THE RECOMBINANT ADENOVIRUS OF FUSION GENE ENCODING HYPER-IL-6 Objective To construct the adenoviral vector that expresses fusion gene encoding Hyper-IL-6 on the basis of successfully amplifying Hyper-IL-6 by PCR. Methods The Hyper-IL-6 gene was coloned to the shuttle plasmid pAdTrack-CMV. The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells. Before transfection, the recombinant adenoviral plasmids were digested with PacI. Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000.Generation of recombinant adenovirus was monitored by GFP expression. Presence of recombinant adenoviruses was further confirmed by PCR. Results The results show that we have successfully cloned the fusion gene encoding Hyper-IL-6 to the shuttle plasmid pAdTrack-CMV. The restriction analysis and the PCR conformed that correct recombinant adenoviral plasmid was constructed. Two days after transfection, the fluorescence was observed in 293 cells. Conclusion The success in construction of the adenoviral vector make it possible to study further on Hyper-IL-6 mediated gene therapy of liver related diseaes.

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