Anti-myoglobin antibody, and kit and method for detecting myoglobin
A myoglobin and antibody technology, applied in chemical instruments and methods, anti-animal/human immunoglobulin, biological testing, etc.
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Embodiment 1
[0111] 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.
[0112] 1 Construction of recombinant plasmids
[0113] (1) Antibody gene preparation
[0114] The mRNA was extracted from the hybridoma cell line (5G9) of the anti-myoglobin monoclonal antibody, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after adding A with rTaq DNA polymerase, and transformed In the DH5α competent cells, after the growth of colonies, four heavy chain and light chain gene clones were respectively taken and sent to a gene sequencing company for sequencing.
[0115] (2) ...
Embodiment 2
[0135] Antibody performance testing
[0136] (1) Activity detection of the antibody and its mutants in Example 1
[0137] Further analysis, the heavy chain variable region of the myoglobin monoclonal antibody (WT) of Example 1 is shown in SEQ ID NO: 12, wherein, wherein, the amino acid sequence of each complementarity determining region is as follows:
[0138] CDR1-VH: G-Y-T-Y(X1)-T-E(X2)-F-S-V(X3)-H;
[0139] CDR2-VH: W-I(X1)-N-S(X2)-E-T-S-E(X3)-P-T-Y-G(X4)-D-D-F;
[0140] CDR3-VH: A-R-R-A(X1)-Y-Y-L(X2)-F-G;
[0141] Its light chain variable region is shown in SEQ ID NO: 11, wherein the amino acid sequence of each complementarity determining region of the light chain is as follows:
[0142] CDR1-VL: R(X1)-A-S-K(X2)-S-V-D-F(X3)-N(X4)-G-D-S-Y;
[0143] CDR2-VL: A-A-S-Q(X1)-R-E(X2)-S;
[0144] CDR3-VL: Q-H(X1)-S-N-E-E(X2)-P-Y.
[0145] On the basis of the myoglobin monoclonal antibody in Example 1, mutations were carried out at sites related to antibody activity in the com...
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