Binding protein of NS1 protein
A technology of binding proteins and proteins, applied in the field of binding proteins of NS1 protein, can solve the problems of unstable production, large influence of individual mice, large batch differences, etc., and achieves low production difficulty, stable antibody function, high sensitivity and specificity. sexual effect
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Embodiment 1
[0148] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. SMARTERTM RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting Anti-Dengue NS1 3D5 monoclonal antibody is an existing hybridoma cell line of Faipeng Biological Co., Ltd., which is recovered for use.
[0149] 1.1 Primers
[0150] Amplify Heavy Chain and Light Chain 5'RACE Primers:
[0151] SMARTER II A Oligonucleotide:
[0152] 5'-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3';
[0153] 5'-RACE CDS Primer (5'-CDS): 5'-(T) 25 VN-3'(N=A,C,G,orT; V=A,G,orC);
[0154] Universal Primer A Mix (UPM):
[0155] 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';
[0156] Nested Univers...
Embodiment 2
[0171] 1. Activity identification of expression supernatant binding protein
[0172] The plasmid was diluted to 400ng / ml with ultrapure water, and the CHO cells were adjusted to 1.43×10 7 cells / mL in a centrifuge tube, mix 100 μL plasmid with 700 μL cells, transfer to an electroporation cup, electroporation, transfer to 10 mL CD CHO AGT medium, and culture in a shaker at 37°C (8% CO 2 , Amplitude 150); samples were taken every day to detect the cell viability, when the cell viability was lower than 50%, the cell culture supernatant was centrifuged to obtain a protein sample.
[0173] To detect the antibody activity after the mutation, dilute goat anti-mouse IgG 1 μg / mL in the coating solution to coat the microplate, 100 μL per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry; add blocking solution (20%BSA+80%PBS), 120μL per well, 37℃, 1h, pat dry; add diluted DN monoclonal antibody, 100μL / well, 37℃, 60min; shake off the liquid in the plate, p...
Embodiment 3
[0180] Although the antibody obtained in Example 2 (with sequences such as light chain and heavy chain shown in SEQ ID NO: 11 and 12) has the ability to bind NS1 protein, its affinity and antibody activity are not ideal, so the applicant's research on this antibody The light chain CDRs and heavy chain CDRs were mutated.
[0181] After analysis, the complementarity determining region of the heavy chain:
[0182] CDR-VH1 is G-F-N-I-K-E(X1)-Y-Y-V(X2)-H;
[0183] CDR-VH2 is W-I-D-P-D(X1)-N-G-K-T-L(X2)-Y-D-P-K-V(X3)-Q-D;
[0184] CDR-VH3 is V-T(X1)-A-Y-V(X2)-R-F-V-Y;
[0185] Complementarity-determining regions of the light chain:
[0186] CDR-VL1 is S-A-S-S(X1)-S-V-K(X2)-Y-M-Y;
[0187] CDR-VL2 is I-Y-E(X1)-T-S-N-V(X2)-A-S-G-F(X3)-P;
[0188] CDR-VL3 is Q-Q(X1)-S-S-T(X2)-P-R-T-F.
[0189] Among them, X1, X2, and X3 are mutation sites.
[0190] After the mutation, the method provided in Example 2 was used to detect the antibody activity, and some results were as follows:
...
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