Hepatitis B virus pre-s1 antigen-specific single-chain antibody and its application
A single-chain antibody and light chain technology, applied in the direction of antibodies, applications, antiviral agents, etc., can solve the problems of product batch differences, long cycle time, human heterogeneity, etc.
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[0022] In the present invention, the antibody can also be linked with a tag to facilitate separation and purification, and the tag can be a variety of common protein tags. According to another preferred embodiment of the present invention, the sequence of the single-chain antibody consists of SEQ ID NO: 1 and the V5 tag and His tag sequentially connected to the C-terminus of SEQ ID NO: 1 (as shown in SEQ ID NO: 3).
[0023] The present invention also provides a gene encoding the above-mentioned single-chain antibody.
[0024] According to a preferred embodiment of the present invention, the sequence of the gene may be as shown in the 1-786th base of SEQ ID NO: 2, or as shown in SEQ ID NO: 2 (containing the V5 tag coding sequence and His tag coding sequence) shown. In this preferred embodiment, positions 1-351 of SEQ ID NO: 2 (capitalized part at the 5' end) show the light chain variable region coding sequence; positions 352-387 of SEQ ID NO: 2 (middle The uppercase underline...
Embodiment 1
[0040] This example is used to illustrate the preparation of a large-capacity fully human antibody library.
[0041] (1) Isolation of human peripheral blood mononuclear cells
[0042] Collect 200 anticoagulated peripheral blood samples from healthy people, each about 1 mL, and use human peripheral blood lymphocyte separation medium (purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd.) to separate lymphocytes.
[0043] ① Add the same volume of separation liquid as the anticoagulant blood into the centrifuge tube, then carefully draw the blood sample with a pipette and slowly add it above the liquid level of the separation liquid, centrifuge at 700g for 30 min.
[0044] ② Use a pipette gun to carefully transfer the ring-shaped milky white mononuclear cell layer of the second layer in the centrifuge tube to another centrifuge tube.
[0045] ③Add 10mL PBS to wash, centrifuge at 250g for 10min, discard the supernatant, repeat this step twice.
[0046] ④Resuspend the cell...
Embodiment 2
[0059]This example is used to illustrate the preparation of HBV preS1 (aa91-107) (the amino acid sequence of HBV preS1 is shown in NCBI protein database: CAA66700.1) specific ScFv
[0060] (1) Panning of HBV preS1 (aa91-107) epitope peptide-specific ScFv
[0061] ① Coat the immunotube with HBV preS1 (aa91-107) epitope peptide, and let it stand or shake overnight at 4°C. Add cultured phages after blocking and washing, wash to remove unbound components after incubation, dissolve specifically bound phages with 0.1M HCl, inoculate in competent Escherichia coli OMNImax culture, culture overnight at 37°C, collect culture supernatant, apply PEG -NaCl precipitation, after centrifugation, resuspend the pellet with PBS, and use this as the sample for the next round of panning. A total of 4 rounds of panning were performed, and the panned products were plated (2YT / Tet / carb) for culture.
[0062] ②Apply preS1 recombinant protein (from plasmid pET22b-preS1 to transform Escherichia coli BL...
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