Human Anti-hbsag Dsfv Antibody Targeting Interferon Composite Gene Cloning And Expression

Following the animal antiserum and rodent monoclonal antibodies, human engineering anti-body is the third generation of antibody which overcomes the shortcomings of antigenicity among species in antiserum and monoclonal antibody and has a potential therapeutic use in human diseases. Human immunoglobulin combinatorial library was generated by using phage surface display expression system, from which phage antibodies (Fab fragments) against HBsAg were screened. The mutated gene of dsFv including the VH and VL of antibody against HBsAg was constructed by PCR-based point mutagenesis method. The mutated genes of VH and VL-IFNa were respectively cloned into two different plasmids, and expressed well after co-transfec-tion. The whole idea of my research is to combine genes VL-IFNa and VH of the antibody together with F2A in an expression plasmid with an open reading frame (ORF) and express the fusion gene in the same cell. The expression of the recombinant expression plasmid include VH and VL of the antibody, and IFN-α, which could be targeting HBsAg and also sublime the effect of IFNα.Firstly, VH gene of human HBsAg dsFv antibody and the DNA fragment with positive charge were constructed by overlapping extension PCR to change the electricity of the antibody. And the combination of VL and human IFN-a gene were connected with the 6×His tab to detect and purify easily. The former research had proved that the DNA fragment could express a cationic peptide which was fused with the recombinant protein, so these protein could combine with DNA through electrostatic interaction. Then, the combination genes VL-IFNa and VH of the antibody linked with F2A with an open reading frame into a same a transitional vector pCI-GPI.Secondely, the recombinant genes of VL-IFN-αand VH in the transitional vector pCI-GPI were inserted into the eukaryotic mammalian expression vector pEE14.1 and the baculovirus expression vector pAcGP67A respectively. The recombinant plasmid were transfected to the CHO-K1 cells and the Sf9 cells in different ways, and the expressions were detected by RT-PCR, ElISA and Western Blot and so on. Otherwise, the protein in the supernatant of the two expression systerms was purified and detectded too.In conclusion, the construction of human anti-HBs dsFv IFNαfusion gene and expression of it in the mammalian expression system and insect cell/baculovirus expression system make it possible to carry out further studies against HBsAg and clinical application on targeted therapy to HBV.