Anti-h7n9 fully human monoclonal antibody 6e9 and its preparation method and application
A monoclonal antibody, fully human technology, applied in the field of immunology, can solve the problems of no effective treatment and rimantadine resistance
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Embodiment 1
[0043] (1) Construction of NTH-3T3 cell line stably expressing CD40L
[0044] 3T3-CD40L feeder cells were established using lentivirus. The lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 generations, infected with lentivirus, continued to be cultured and passed 3 times. Use a flow cytometer to sort the cells whose FITC fluorescence intensity is near the MFI, and add them back to the culture flask at 37°C, 5% CO 2 Cultivate and detect in the incubator, and the test results are as follows: figure 1 As shown, 3T3 cells expressing CD40L and 3T3 cells transfected with empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC, and then analyzed by flow cytometry. It was found that all 3T3-CD40L feeder cells expressed CD40L. When the cells grow to 80%-90%, digest and collect the cells at a concentration of 1×10...
Embodiment 2
[0066] Example 2 Cloning, recombination and expression of humanized monoclonal antibody 6E9 gene
[0067] The B cells obtained in Example 1 capable of secreting the antibody 6E9 binding to the H7N9 virus were lysed, and the lysate was taken for reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene. The heavy chain and light chain genes of the antibody were cloned using cDNA as a template, and recombinantly expressed and purified in eukaryotic 293F or HEK293 cells.
[0068] specifically:
[0069] (1) Transfer the B cell liquid to a 96-well plate (Eppendorf, 030133366).
[0070] (2) Reverse transcription system: 150ng random primer (invitrogen, 48190-011), 0.5ul 10mM dNTP (Invitrogen, 18427-088), 1μl 0.1M DTT (Invitrogen, 18080-044), 0.5% v / v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), add DEPC water to 14μl / well.
[0071] (3) Revers...
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