Bispecific antibody for hepatitis B surface protein, and use thereof
A bispecific antibody and surface protein technology, which is applied in the direction of antibodies, antiviral immunoglobulins, applications, etc., can solve problems such as limiting the development process, high development costs, and inability to meet clinical needs, achieving strong synergistic effects and low cost. Immunogenic effects
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Embodiment 1
[0032] Example 1: Bispecific Antibody A 3 B 5 - Preparation of BsAb
[0033] 1. Cloning of antibody light chain constant region and heavy chain constant region genes and construction of their vectors
[0034] Isolate healthy human lymphocytes with lymphocyte separation medium, extract total RNA with Trizol reagent, design primers according to the sequences reported in literature [8] and literature [9], and use QIAGEN OneStep RT-PCRKit to amplify the heavy chain and light chain constant regions of human antibodies Gene. The heavy chain and light chain constant region genes of the human antibody were respectively connected to the expression vector AbVec plasmid, and the antibody heavy chain constant region nucleotide vector IgG-AbVec plasmid and the antibody light chain constant region nucleotide vector Igκ-AbVec were respectively constructed Plasmid, after sequencing verification, it was confirmed that the correct clone was obtained. Wherein, the nucleotide sequence and ami...
Embodiment 2
[0044] Example 2, bispecific antibody A 3 B 5 -Specificity of BsAb
[0045] Detection of bispecific antibody A by Elisa method 3 B 5 - the ability of the BsAb to specifically bind the HBsAg protein.
[0046] The recombinant HBsAg protein and the control protein cIg were coated on the ELISA plate, and after being blocked by the blocking solution, the blank group, different concentrations of the control protein cIg, and different concentrations of expressed and purified A 3 D. 5 Antibody incubation, adding Goatanti-humankappa-HRP after washing the plate, TMB chromogenic solution for color development, microplate reader reads at 450nm wavelength.
[0047] Draw according to antibody concentration and corresponding OD value figure 1 ,Depend on figure 1The analysis shows that with the increase of the antibody concentration, the corresponding reading value of the HBsAg protein group is also correspondingly increased. However, the reading value of the coating control protein g...
Embodiment 3
[0048] Example 3, bispecific antibody A 3 B 5 - BsAb binding epitope identification
[0049] Combining the antigenic epitope region of hepatitis B surface antigen (HBsAg) with neutralizing activity reported in previous literature, a biotin-labeled short peptide of hepatitis B surface antigen was synthesized to measure the binding of fully human hepatitis B surface protein monoclonal antibody on HBsAg area. Such as figure 2 As shown in the HBsAg pattern diagram, the synthetic four biotin-labeled HBsAg short peptide P 1 (aa:104-120), P 2 (aa:121-137), P 3 (aa:139-148), P 4 (aa:149-163), P 1 -P 4 The amino acid sequence is shown in SEQ ID NO.9-SEQ ID NO.12. Among them, the antigenic short peptide P 1 and P 4 is a linear structure, P 2 and P 3 ring structure [10] .
[0050] Preliminary experiments showed that binding to the conformational epitope segment (P 2 and P 3 The monoclonal antibody neutralizing HBV activity is better than the linear epitope segment (P 1...
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