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Human anti- HBsAg dsFv antibody targeting interferon composite gene cloning and expression

Author: JiangLe
Tutor: GuoBingRan；YuJiYun
School: Qufu Normal University
Course: Zoology
Keywords: Hepatitis B virus surface antigen Disulfide stabilized Fv antibody Interferon alpha Mammalian expression systems Insect cell / baculovirus expression system Plasmid Construction Gene expression
CLC: R512.62
Type: Master's thesis
Year: 2010
Downloads: 46
Quote: 0
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Abstract


Antibody technology has undergone serum polyclonal antibodies, monoclonal antibodies stage after the development of the third generation of antibodies - genetically engineered antibodies. In preliminary studies to human anti HBsAg gene engineering antibody, PCR site-directed mutagenesis to obtain disulfide-stabilized dsFv (disufide-stabilized Fv fragments) antibody phage display library screening, antibody light chain with interferon alpha fusion genes and heavy chain genes were cloned into plasmid pCI-neo and pCdhfrl co-transfected cells in order to obtain expression. This study was to further explore ways to improve on this basis, the whole idea - interferon alpha fusion genes and heavy chain gene by cutting itself peptide F2A connections under the same open reading frame of the human anti-HBsAg dsFv antibody light chain, to achieve antibody series expression of the light and heavy chains in the same plasmid. First constructed in the transition vectors PCI-GPI antibody targeting interferon composite gene: dsFv VH of the antibody heavy-chain gene 3 'end of the integration period of the positively charged DNA load region gene by overlap extension PCR method, the target protein positively charge, combined with a negatively charged plasmid DNA; 3 'end connected to the 6 × His tag fusion gene and the antibody light chain - interferon alpha, in order to facilitate the detection and purification of the expressed protein. Cutting through itself and the light chain peptide F2A the human anti-HBsAg antibody dsFv composite gene and composite heavy chain gene was ligated placed in the same open reading frame, and light, and the composite gene of the heavy chain connection can be implemented in the the transition vectors PCI-GPI restructuring plasmids pCIGPI-dsFvαpr. Next, choose the mammalian expression system and insect cell / baculovirus expression system for recombinant complex gene expression antibody targeting interferon. On the basis of building the pCIGPI-dsFvαpr | antibody targeting interferon composite gene by restriction enzyme digestion and PCR and cloned into the eukaryotic expression vector pEE14.1 and the baculovirus expression system transfer vector pAcGP67A, to obtain the antibody-containing targeting interferon the composite gene recombinant to the expression plasmid pEE14.1-dsFvαpr and pAcGP67A-dsFvαpr, and verify its expression in mammalian CHO-K1 cells and insect cells Sf9. In addition, we also mammalian expression system and insect cell / baculovirus expression system to express the target protein in the supernatant were purified, and its preliminary validation. In short, containing antibodies targeting interferon recombinant gene recombinant plasmid attempt to build its expression in mammalian expression systems and insect cell / baculovirus expression system for subsequent antibody targeting interferon protein binding HBV therapeutic DNA vaccine anti-hepatitis B targeted nanoparticle drug development laid the foundation, but also for the HBV immune gene therapy offers a new technology strategy.

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CLC: > Medicine, health > Internal Medicine > Infectious disease > Viral infections > Viral Hepatitis > Hepatitis B
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