Construction Of Bispecific Antibodies Against Two Different Epitopes On NP And Glycoprotein G2 Of Hantaan Virus And Expression In Insect Cells

Hemorrhagic fever with renal syndrome(HFRS), which is caused by hantavirus, is an acute infectious disease characterized by fever, vascular hemorrhage and kidney dysfunction. HFRS is more severely in China. About 50,000-100,000 case of HFRS are reported yearly and HFRS has a high death toll. Until now, there are still no effective therapeutical drugs directed to HFRS.The development of mouse monoclonal antibodies(mAb) technology has allowed generation of specific antibodies and thus provides a new approach to antiviral therapy. However, human anti-mouse antibody (HAMA) is provoked by the application of mouse mAb in the clinical therapy, which not only probably depress curative effect but also can result in severely allergies. How to depress the immunogenicity of the mouse mAb is the urgent problem which need to be solved in the fundamental research on theclinical application of the mouse mAb. Micromolecule antibodies, which are the new generation antibodies founded on genetic engineering techniques, provide a useful way for the application of the mouse mAb. Micromolecule antibodies can hold the immunologic competence of the parental antibody, meanwhile, they have many good qualities such as lower immunogenicity and powerful tissue penetration. We prepare micromolecule antibodies by genetic engineering techniques and degress its immunogenicity as far as possible which is one of the effective approaches to overcome the disadvantage of the mouse mAb.In this study, expression vectors carrying chimeric gene of the bispecific antibody against two different epitopes on NP and glycoprotein G2 of HTNV were constructed and expressed in E.coli. JM109 and insect cells, respectively. The immunological activities of the expressed products were identified. This research may serve as a new generation drugs for the therapy of HFRS.Two heterogeneous single chain Fv genes were constructed by fusing Vh gene of one mouse mAb 1A8 with Vl gene of another mAb 3G1 and vice versa. The human k light chain constant region gene was fused in-frame at the C-terminus of 1A8 VL gene. The two heterogeneous ScFv genes were cloned into pComb3 and transformed into E.coli. JM109 and induced with IPTG. The lysate of the cells could be detected that having activity of binding HTNV nucleoprotein by the indirect immunofluorescence and ELISA test. Meanwhile, a recombinant baculocirus expression vector ScFv-pFBD-ScFv' carrying chimeric genes of a bispecific antibody against HTNV was constructed. The genes were inserted into shuttle plasmid in E.coli. DH10Bac...