Human anti-vascular endothelial cell growth factor antibody and application thereof
An antibody, whole antibody technology, applied in the direction of anti-growth factor immunoglobulin, application, antibody, etc.
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Embodiment 1
[0056] Example 1 Large-scale screening of large-capacity phage antibody library
[0057] 1. Materials and methods:
[0058] 1. Materials: A large-capacity fully synthetic phage single-chain antibody library was constructed by the Academy of Military Medical Sciences of the Chinese People's Liberation Army (ZL200910091261.8), with a library capacity of 1.35×10 10 . The antigen for screening the antibody library was recombinant VEGF165 expressed by HEK293T cells (concentration: 1 mg / ML), the strain was XLI-Blue (Stratagene, USA); the phage used was M13KO7 (Invitrogen, USA). The eukaryotic expression vectors pABG1, pABκ and pABL are preserved in this laboratory, and the vector information can be found in ( Figure 7A , Figure 7B , Figure 7C ), mammalian HEK293T cells were purchased from Invitrogen.
[0059] 2. Method
[0060] 2.1 Preparation of recombinant protein antigen VEGF165:
[0061] The VEGF165 gene (gifted by Dr. Minglei Shi from the Institute of Bioengineering, ...
Embodiment 2
[0081] Example 2 Computer-aided design of in vitro affinity maturation of anti-VEGF antibody VA6
[0082] 1. Experimental method
[0083] 1.1 Construction of 3D structure model of VA6-VEGF complex
[0084] 1.1.1 Database search for amino acid sequences
[0085] Using the variable region sequences of the light and heavy chains of antibody VA6 as probes, in the latest protein structure database (PDB), the Fasta3.0 program was used to search for sequences, and found highly homologous to the light and heavy chains of VA6, respectively. Reference protein, the homology between the reference protein sequence and the antibody sequence obtained by the search is more than 40%, and the homology between the complex and ICZ8\VEGF complex crystallization reaches 94% after structural superposition, which meets the requirements for obtaining a high-precision structural model .
[0086] 1.1.2 Homology modeling of 3D structures
[0087] Homology modeling was carried out using the program mo...
Embodiment 3
[0107] Example 3 Construction and screening of VA6-9B light chain Fab recombinant phage antibody library
[0108] 1. Materials and methods
[0109] 1. Construction of Phage Fab Antibody Library
[0110] In order to obtain mutant antibodies with higher affinity, the heavy chain of VA6-9B was selected as the object, and the Vκ light chain gene library was constructed through the chain exchange strategy, which was combined with the heavy chain of VA6-9B to construct the light chain mutation Antibody library H9B-Vκ. References for light chain gene library Vκ gene design (Knappik A, Ge LM, Honegger A, et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 2000, 296: 57- 86; Dewey's doctoral dissertation: Academy of Military Medical Sciences, 2006), the framework gene sequence of Vκ is shown in SEQ ID No.35. The amino acid sequences of its CDR1 region and CDR2 region are RAS...
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