Anti-influenza A virus antibody, detection kit and preparation method
An influenza A virus, antibody technology, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as specificity and sensitivity defects
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Embodiment 1
[0104] 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. BD SMART TM 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.
[0105] 1 Construction of recombinant plasmids
[0106] (1) Antibody gene preparation
[0107] The mRNA was extracted from the hybridoma cell line (4G2) secreting anti-influenza A virus antigen antibody, and the DNA product was obtained by RT-PCR method, and the product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, Transform into DH5α competent cells, and after the colonies grow out, take 4 clones of Heavy Chain and Light Chain gene clones and send them to a gene sequencing company for sequencing...
Embodiment 2
[0128] Antibody performance testing
[0129] (1) Activity detection of the antibody and its mutants in Example 1
[0130] Analysis of the antibody (WT) sequence of Example 1, its heavy chain variable region is shown in SEQ ID NO: 12, wherein, wherein, the amino acid sequence of each complementarity determining region on the heavy chain variable region is as follows:
[0131] CDR-VH1: G-F-S-I(X1)-T-A-Y(X2)-G-L(X3)-H;
[0132] CDR-VH2: LI(X1)-W-G(X2)-G-G-S-T-N-Y-N-A(X3)-T-L(X4)-M-S;
[0133] CDR-VH3: A-K(X1)-E-V(X2)-T-G-R-M;
[0134] Its light chain variable region is shown in SEQ ID NO: 11, wherein the amino acid sequence of each complementarity determining region on the light chain variable region is as follows:
[0135] CDR1-VL: K-A-S-Q-D-L(X1)-N-Q(X2)-Y-I(X3)-S;
[0136] CDR-VL2: R-A-N-R-I(X1)-L(X2)-D;
[0137] CDR-VL3: I(X1)-Q-Y-N(X2)-E-F-P-Y.
[0138] On the basis of the anti-influenza A virus antibody (WT) in Example 1, mutations were carried out at sites related to...
Embodiment 3
[0169] Application of Antibody in Colloidal Gold Detection
[0170] 1 Preparation of colloidal gold detection test paper
[0171] (1) Preparation of nitrocellulose membrane
[0172] Preparation of coating buffer: 6% methanol, 0.01M pH7.2 PBS buffer as coating buffer, filtered through 0.22 μm membrane, set at 4°C for later use, valid for one week. 1000ml 6% Methanol 0.01M pH 7.2PBS Buffer Recipe: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60ml, distilled deionized water to 1000ml.
[0173] Preparation of nitrocellulose membrane: Dilute the purified antibody to 1-5 mg / ml with coating buffer, adjust the machine, draw the T line, which is the detection line, and the T line is close to the end of the gold standard pad and away from the end of the gold standard pad. About 5mm; Dilute the goat anti-mouse IgG antibody to 1-5mg / ml with coating buffer, adjust the machine, draw line C, which is the control line, and line C is close to the absorbent pad...
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