Heavy chain and light chain variable region gene of human B lymphocyte stimulation factor monoclonal antibody and its application
A technology of B lymphocytes and monoclonal antibodies, applied in the direction of anti-cytokine/lymphokine/interferon immunoglobulin, application, genetic engineering, etc., can solve the problems of cell line instability, high cost, easy loss, etc., to achieve Not easy to save, high production cost, good for industrial production
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Embodiment 1
[0033] Example 1: The variable region gene of antibody isolated from BLyS monoclonal cell line ABL-1 is collected and grows well, the hybridoma cell line ABL-110 that can secrete BLyS monoclonal antibody 7 One (constructed by our laboratory, according to "Current protocol in immunology" Production of Monoclonal Antibodies), total RNA was extracted with TRIZOL reagent.
[0034] RT-PCR catch VH gene:
[0035] With reference to the method provided by Promega (Access RT-PCR System and Access RT-PCR Introductory System): one-step RT-PCR kits were used to amplify the VH gene respectively:
[0036] The VH gene uses primer VH Back (5'-AGGTSMARCTGCAGSAGTCWGG-3') and primer VH For (5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'),
[0037] The RT-PCR reaction is a total of 50 μl system: 1 μg of total RNA, 10 μl of 5×AMV / Tf1 reaction buffer, 5 μl of primers VH Back and VH For at a concentration of 10 pmol, 1 μl of dNTP at a concentration of 500 uM, and 3 mM MgSO 4 2 μl; 1 μl of 5u AMV reverse...
Embodiment 2
[0047] Example 2: Assembly of scFv genes
[0048] To obtain scFv, add a flexible linker between VH and VL (G 4 S) 3 , in order to prevent the introduction of mutations during the amplification process, we use the high-fidelity enzyme Pyrobest (Dalian Bao Biology). The whole assembly schematic diagram is figure 2 , the result is as figure 1 .
[0049] The first round of PCR reaction to amplify the VH:
[0050] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; the concentration of 10 μM primer VH P 1 and VH P 2 2.5 μl: 4 μl dNTPs; 35 μl HO 2 O; High-fidelity Pyrobest enzyme 0.5 μl. Denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 26 cycles; extension at 72°C for 5min.
[0051] The second round of PCR reaction amplifies VL:
[0052] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; primer VL P at a concentration of 10 μM 1 and VLP 2 2.5 μl: 4 ...
Embodiment 3
[0056] Example 3: Enzyme digestion and connection of scFv gene
[0057] Double digestion of pET28a:
[0058] EcoRI and HindIII are Takara products. 2 μl of 10×M reaction buffer; 10 μl of vector pET28a; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours. :
[0059] The third round of amplification product in double enzyme digestion embodiment 2:
[0060] 2 μl of 10×M reaction buffer; 10 μl of the third-round PCR product; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours.
[0061] 1% agarose gel electrophoresis, and the product was recovered by tapping the gel.
[0062] Link reaction:
[0063] T4 ligase is a product of Takara. 2 μl of ligation reaction buffer H 2 O; 1 μl each of the products of the last round of enzyme digestion; 16 μl H 2 O. React at 16°C for 12 hours.
[0064] Take 10 μl of the product to transform into competent DH5α. Plate the plate, pick a single clone the next day, identify it by colony PCR, and s...
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