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Binding protein of NS1 protein

A protein-binding protein technology, which is applied in the field of NS1 protein-binding protein, can solve problems such as unstable production, large influence on individual mice, and large batch-to-batch variation, achieving high sensitivity and specificity, stable antibody function, and difficult production small effect

Active Publication Date: 2019-01-04
DONGGUAN PENGZHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the hybridoma production method adopts mouse peritoneal induction, it is particularly affected by the individual mice, the production is unstable, the batch-to-batch difference is large, and the purification of mouse autoantibodies is difficult.

Method used

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  • Binding protein of NS1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] 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 7D9 monoclonal antibody is an existing hybridoma cell line of Faipeng Biological Co., Ltd., which is recovered for use.

[0156] 1.1 Primers

[0157] Amplify Heavy Chain and Light Chain 5'RACE Primers:

[0158] SMARTER II A Oligonucleotide:

[0159] 5'-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3';

[0160] 5'-RACE CDS Primer (5'-CDS): 5'-(T) 25 VN-3'(N=A,C,G,orT; V=A,G,orC);

[0161] Universal Primer A Mix (UPM):

[0162] 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGA GT-3';

[0163] Nested Univer...

Embodiment 2

[0178] 1. Activity identification of expression supernatant binding protein

[0179] The plasmid was diluted to 400ng / ml with ultrapure water, and the CHO cells were adjusted to 1.43×10 7cells / 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.

[0180] Antigen DN-II-Ag# (Fei Peng Biological Products) was diluted 1000 times with CB, added 100 μL to each well of polystyrene enzyme block, and stayed overnight at 4°C; the next day, washed twice with washing solution PBST and patted dry; solution (20% BSA+80% PBS), 120 μL per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100μL / well, 37°C, 30min (partial supernatant 1h); wash with washing sol...

Embodiment 3

[0187] 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.

[0188] After analysis, the complementarity determining region of the heavy chain:

[0189] CDR-VH1 is A-S-G-Y-T(X1)-F-T-G-F(X2)-Y-M-H;

[0190] CDR-VH2 is R-V-N-P-Q(X1)-N-G-G-S(X2)-S-Y-N-Q-K-F-V(X3)-G;

[0191] CDR-VH3 is A-Q(X1)-E-G-V(X2)-H-Y-D-R-A;

[0192] Complementarity-determining regions of the light chain:

[0193] CDR-VL1 is Q-S-L-L-Y-S(X1)-S-N-N(X2)-K-N-S-L-A;

[0194] CDR-VL2 is W-A-S-T-Q(X1)-E-S;

[0195] CDR-VL3 is Q-Q-Y-F(X1)-T-Y-A(X2)-Y-T.

[0196] Among them, X1, X2, and X3 are mutation sites.

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PUM

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Abstract

The invention provides an isolated binding protein having an antigen binding structural domain which is bound with NS1 protein. The isolated binding protein comprises specific heavy chain CDR and light chain CDR, can recognize and bind with the NS1 protein specifically, has high flexibility and specificity, and can realize detection on dengue fever virus. The way of utilizing mouse peritoneal induced hybridoma to produce the binding protein is omitted, and thus, production difficulty of the binding protein is reduced while antibody function thereof is much stable.

Description

technical field [0001] The invention relates to the fields of biotechnology and medical technology, in particular to a binding protein of NS1 protein. Background technique [0002] Dengue fever (dengue fever, DF) is an acute mosquito-borne infectious disease caused by four serotypes of viruses (DENV-1, DENV-2, DENV-3, DENV-4), mainly transmitted by Aedes aegypti and Aedes albopictus . DF is an arbovirus disease with the widest distribution, the most incidence, and great harm. It is widely prevalent in more than 100 countries and regions in tropical and subtropical Africa, America, Southeast Asia and the Western Pacific. [0003] Clinically, DF is a severe flu-like illness. The main manifestations are sudden onset, high fever, severe headache, retroorbital pain, muscle and joint pain, and may be accompanied by rash, lymphadenoma, and leukopenia. This type of disease is generally called classical dengue fever, which spreads rapidly and can cause large-scale epidemics. Duri...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569
CPCG01N33/56983C07K16/10C07K2317/92C07K2317/56C07K2317/565G01N2469/10C07K2317/94C07K16/1081G01N2333/185Y02A50/30C07K2317/20C07K2317/52C07K2317/567
Inventor 崔鹏何志强孟媛钟冬梅
Owner DONGGUAN PENGZHI BIOTECH CO LTD
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