Anti-influenza B virus antibody, preparation method thereof and detection kit
A type of influenza B virus and antibody technology, applied in antiviral immunoglobulins, measuring devices, using vectors to introduce foreign genetic material, etc., can solve the problems of antibody specificity and sensitivity defects
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Embodiment 1
[0106] 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.
[0107] 1 Construction of recombinant plasmids
[0108] (1) Antibody gene preparation
[0109] The mRNA was extracted from the hybridoma cell line secreting anti-influenza B virus antibody, and the DNA product was obtained by RT-PCR method. The product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase, and transformed into DH5α. In the state cells, the Heavy Chain and Light Chain gene clones were obtained after the colonies grew out, and 4 clones each were sent to a gene sequencing company for sequenci...
Embodiment 2
[0130] Antibody performance testing
[0131] (1) Activity detection of the antibody and its mutants in Example 1
[0132] Analysis of the antibody (WT) sequence of Example 1, its heavy chain variable region is shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region is as follows:
[0133] CDR-VH1: G-F-S(X1)-F-S-S-F(X2)-T-F(X3)-S;
[0134] CDR-VH2: T-V(X1)-S-S-G-G-S-Y-S(X2)-Y-Y-P-D-S-I(X3)-K-G;
[0135] CDR-VH3: T-K(X1)-H-L(X2)-T-T(X3)-A-S-Y;
[0136] 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:
[0137] CDR1-VL: R-A-S-E(X1)-N-L(X2)-Y-T-S-V(X3)-A;
[0138] CDR-VL2: Y-G(X1)-A-T-N-I(X2)-A-D;
[0139] CDR-VL3: Q-Q(X1)-F-W-A(X2)-T-P-W-T.
[0140] On the basis of the anti-influenza B virus antibody (WT) in Example 1, mutations were carried out at sites related to ...
Embodiment 3
[0171] Application of Antibody in Colloidal Gold Detection
[0172] 1 Preparation of colloidal gold detection test paper
[0173] (1) Preparation of nitrocellulose membrane
[0174] 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 formulation: 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.
[0175] Preparation of nitrocellulose membrane: Dilute the purified antibodies in Table 3 and Table 5 to 1-5 mg / ml with coating buffer respectively, adjust the machine, draw the T line, which is the detection line, and the T line is close to the gold standard The end of the pad is about 5mm away from the pad end of the gold standard; dilute the goat anti-mouse IgG antibody to 1-5 mg / ml with the coating buffer, adjust the machine, and draw the line C, which is ...
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