TBAB humanized monoclonal antibody recombinant vector, recombinant antibody and preparation method of recombinant antibody
A technology for cloning antibodies and recombinant vectors, applied in the biological field, can solve the problems of poor stability and low yield of TBAB antibodies, achieve high antibody activity, broad application prospects, and meet the needs of scientific research and rapid detection
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Embodiment 1
[0029] Example 1 Construction of pCHO1.0-TBAB expression plasmid
[0030] Construction of stable expression cell lines.
[0031] Methods as below:
[0032] 1. Separation of plasma cells: collect blood from patients with Graves' disease, use density gradient centrifugation to separate plasma cells, extract RNA, and use RT-PCR to obtain cDNA.
[0033] 2. As shown in SEQ ID NO:1 and SEQ ID NO:2.
[0034] 3. Using specially designed upstream and downstream primers to PCR clone the heavy chain variable region (VH) and light chain variable region (VL) of the hybridoma cell.
[0035] Wherein the heavy chain (VH) primer sequence is shown in SEQ ID NO:3 and SEQ ID NO:4:
[0036] Upstream primer: 5' cctag caaatgcagctggtgcag3' (SEQ ID NO: 3);
[0037] Downstream primer: 5' gtatac tgaggagacggtgaccag 3' (SEQ ID NO: 4).
[0038] The light chain (VL) primer sequence is shown in SEQ ID NO:5 and SEQ ID NO:6::
[0039] Upstream primer: 5' gatatc ctgcctgtgctgactcag 3' (SEQ ID NO: 5);...
Embodiment 2
[0047] Example 2 pCHO1.0-TBAB expression plasmid was introduced into recipient cells to obtain recombinant antibody
[0048] The specific operation steps are as follows:
[0049] (1) co-transfect the eukaryotic expression cell line CHO with the expression vector connecting the heavy chain (VH) and light chain (VL) genes of the intended monoclonal antibody obtained in Example 1;
[0050] Wherein, in the step (1), the eukaryotic expression cell line CHO is a suspension culture, cultured with CD011 complete medium, and the culture condition is 37 ° C, 5% CO 2 , 120rpm; take CHO cells, count with trypan blue, the cell viability is above 98%, and use 0.5-1×10 6 / mL concentration inoculated cells in CD011 complete medium, 37°C, 120rmp, 5% CO 2 to cultivate.
[0051] (2) The day of inoculation is recorded as day 0, and the cell density is counted at about day 3 and day 4. If the cell density is greater than 4×10 6 / mL, add sodium butyrate (purchased from Sangon Bioengineering (Sh...
Embodiment 3
[0055] Example 3 Determination of recombinant antibody protein content
[0056] Every other day, 1 mL of the medium was taken, and the expression of antibody protein was determined by the BCA quantitative method.
[0057] Specific steps are as follows:
[0058] (1) Preparation of protein standards
[0059] a. Take 0.8mL of protein standard preparation solution and add it to a tube of protein standard (20mg BSA), fully dissolve and prepare a 25mg / mL protein standard solution. It can be used immediately after preparation, or can be stored at -20°C for a long time.
[0060] b. Take an appropriate amount of 25mg / mL protein standard and dilute it with culture medium to a final concentration of 0.5mg / mL.
[0061] (2) Preparation of BCA working solution
[0062] According to the number of samples, prepare 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1);
[0063] (3) Determination of protein concentration
[0064] a. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the ...
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