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Expression of Avian Influenza Virus H5N1 HA1 and Mouse IgG Fc Fragment Fusion Protein

Author: HongSuMei
Tutor: LiZiLi；ShiDeShi
School: Huazhong Agricultural University
Course: Preventive Veterinary Medicine
Keywords: FcRn Avian influenza virus H5N1 HA1-Fc fusion protein Expression
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 47
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Abstract

The neonatal Fc receptor (FcRn) are the specific receptors for the identification of the Fc fragment of the immunoglobulin IgG. The most disease infection occurs in the mucous membrane or the invasion of the body by the mucous membrane. FcRn specific binding IgG Fc fragment, active transport of IgG-mediated mucosal epithelial cells, the body's ability to resist pathogens. Therefore can be an antigen and the Fc fragment of the IgG fusion analog IgG across the mucosal surface of the transfer, through the mucosal barrier by FcRn mediated IgG transcytosis pathway transporter antigen. The avian flu virus (Avian Infulenza Virus, AIV) infection of the body through the respiratory tract, with major protective antigen of the H5N1 avian influenza virus hemagglutinin HA1 this study as a model antigen, through a connection (Linker) fragment (amino acid sequence: GSGGGGSGGGGSGS) The mouse IgG2a Fc fragment fusion (HA1-Linker-wFc, abbreviated as HA1-wfc), using the Bac-to-Bac expression system to express the HA1 and IgG2aFc wild-type fragment of the fusion protein (HA1-wFc) and HA1 and IgG2a Fc mutant fragment of the fusion protein (HA1-mFc). FcRn mediated IgG transcytosis pathway transshipment antigen mucosal barrier to further develop in the future and lay a good foundation. The main results achieved in this study are as follows according to the GenBank published on avian flu virus H5N1 HA1 gene (AY830774.1) and mouse IgG2a Fc the gene (V00798.1) sequence primers were designed: HA1-wFc gene cloning, the use of laboratory plasmid pMD18-T-HA1 save pMD18-T-wFc as a template, the avian influenza virus H5N1 HA1 gene and mouse IgG2a wFc fragment gene were amplified, which wFc fragments C1q binding sites mutated lost its binding ability. With a Linker connected to the HA1 and wFc fragment (HA1-wFc), connected with pMD18-T vector of positive recombinant plasmid pMD18-T-HA1-wFc sequenced, except for HA1 gene of wFc of gene (mutation sites) The corresponding gene in GenBank sequence homology was 100%. The of 2. HA1-mFc gene cloning recombinant plasmid pMD18-T-HA1-wFc as a template for mutation by PCR-directed mutagenesis method to wFc FcRn binding key sites, so that the loss of binding capacity with FcRn. Connect the HA1-mFc snippet into pMD18-T vector, positive recombinant plasmid pMD18-T-HA1-mFc sequenced the HA1 the gene and mFc gene (mutation sites except) in GenBank corresponding gene sequence homology was 100%. 3.32a-HA1-wFc and KG-HA1 fusion protein of the original nuclear expression of recombinant plasmid pMD18 T-the HA1-wFc as templates, connect the HA1-wFc snippet vector pET-32a build a reorganization of the original expression plasmid (pET-32a- HA1-wFc), and pGEX-KG-HA1 preserved respectively in the E. coli expression, Western blot analysis and erythrocyte agglutination test showed that the expression the the 32a-HA1-wFc and KG-HA1 fusion protein with good immunological activity. The 4. HA1-wFc and the HAl-mFc fusion protein expression in the baculovirus expression system as a template to of recombinant plasmids pMD18-T-HA1-wFc and pMD18-T-HA1-mFc respectively using enzyme digestion and ligation method HA1- gene fragment wFc and HA1-mFc of connection to the carrier pFASTBAC HTb construct eukaryotic expression plasmid (pFASTBAC HTb.-HA1-wFc and pFASTBAC HTb.-HA1-mFc), and Bac-to-Bac system successfully expressed, Western blot analysis and erythrocyte agglutination tests showed that the expression of the HA1-wFc and HA1-mFc of fusion protein has a good immunological activity.

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