A kind of monoclonal antibody and application thereof for neutralizing Epstein-Barr virus
A technology of Epstein-Barr virus and antibodies, applied in the fields of antibodies, applications, antiviral agents, etc., can solve the problems of human monoclonal antibodies on the market, and achieve the effect of reducing side effects
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Embodiment 1
[0040] Example 1 Preparation of EBV gH / gL recombinant protein
[0041] The gH / gL protein plays an important role in the process of EBV invasion of epithelial cells and B cells. Therefore, the inventor's research team selected gH / gL as the bait protein to screen specific memory B cells in order to obtain neutralizing antibodies that can block EBV infection.
[0042] Then, the genome of the EBV-M81 strain was selected as a template to amplify the sequences of gH and gL respectively, and link them together through a linker to improve the efficiency of protein purification.
[0043] The original sequence of gH before modification:
[0044] ATGCAGTTGCTCTGTGTTTTTTGCCTGGTGTTGCTATGGGAGGTGGGGGCTGCCAGCCTTAGCGA GGTTAAGCTGCACCTGGACATAGAGGGGCATGCTTCGCATTACACCATCCCATGGACCGAACTGATGG CAAAGGTCCCAGGCCTTAGCCCAGAGGCGCTGTGGAGAGAGGCAAATGTCACCGAAGATTTGGCGT CTATGCTTAACCGCTACAAGTTAATTTACAAGACGTCTGGTACCCTTGGTATTGCGCTGGCCGAGCCTG TCGATATCCCTGCTGTCTCTGAAGGATCCATGCAAGTGGATGCATCTAAGGTCCATCCCGGAGTCATTA GCGG...
Embodiment 2
[0085] Example 2 Screening and sequencing of antibodies
[0086] The menory B cells specifically combined with gH / gL protein were screened by flow cytometry.
[0087] (1) Materials and equipment
[0088] Anticoagulant peripheral blood of EBV infected patients; lymphocyte separation fluid; pH7.2 phosphate buffer saline (PBS, Thermo company); PBS-1% (wt / vol) BSA solution; fluorescent antibody CD3-PE-Cy5, CD16- PE-Cy5, CD235a-PE-Cy5, CD19-APC-Cy7, CD20-PE-Cy7, CD38-AF700, IgG-FITC (BD Biosciences); CD27-PB (Biolegend) CD14-PE-Cy5 (Thermo ); anti-his-PE fluorescent antibody (Miltenyi Biotec); His-tag labeled gH / gL protein (gH / gL-his protein); 96-well PCR plate (Axygen, USA); FACSAriaII flow cytometer (BD Corporation).
[0089] (2) Method
[0090] 1) Using Ficoll density gradient centrifugation to separate mononuclear cells (PBMC) from the peripheral blood (heparin anticoagulation) of EBV-infected patients;
[0091] 2) Count PMBC, take 1×10^7 cells and incubate with 100nMgH / gL...
Embodiment 3
[0097] Example 3 Sequencing and Expression Purification of Monoclonal Antibodies
[0098] The single B cell screened in Example 2 was lysed, cDNA was obtained by RT-PCR, and then the heavy chain and light chain specific primers were used to perform nested PCR respectively, and the antibody gene was sequenced.
[0099] Expression and purification of monoclonal antibodies.
[0100] The screened antibody heavy chain variable region upstream and CMV fragment, downstream and human IgG1 constant region and polyA fragment were subjected to overlapping PCR to obtain a fragment that could express the complete heavy chain; the screened antibody light chain variable region upstream Perform overlapping PCR with the CMV fragment, the downstream constant region of the light chain κ / λ, and the poly A fragment to obtain a fragment that can express the complete light chain. These two fragments were then constructed into the PMD18T (Takara) vector. The PMD18T plasmid containing the full-lengt...
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