Preparation of a recombinant canine c-reactive protein and its monoclonal antibody
A reactive protein and recombinant protein technology, applied in the field of canine inflammation early diagnosis and peptides, can solve the problems of poor specificity of monoclonal antibodies, hinder the preparation of monoclonal antibodies, low expression, etc., to improve detection sensitivity and ensure broad detection spectrum. , the effect of improving the expression level
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Embodiment 1
[0017] Example 1: Canine C-reactive protein dominant epitope selection
[0018] Taking canine C-reactive protein as the target antigen, using the biological software DNAssist2.0 to analyze the hydrophilicity and antigenicity of its antigenic epitope sequence, select A dominant antigenic epitope (SEQ ID No: 3) and B dominant antigenic epitope (SEQ ID No: 3) ID No: 4). At the same time, the results of sequence comparison showed that the selected two dominant antigenic epitopes, A and B, had a broad spectrum and were common epitopes for all canine C-reactive protein; and the A and B epitopes had no obvious homology with other protein sequences, Only present in the canine C-reactive protein sequence.
Embodiment 2
[0019] Example 2: Synthesis of polypeptides containing dominant epitopes of canine C-reactive protein
[0020] In order to enhance the activation effect of the selected epitope on the mouse immune system and shorten the preparation time of monoclonal antibodies, two dominant epitope sequences of canine C-reactive protein A and B were chemically synthesized and connected in series, and cysteine was connected at the end (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) to obtain a polypeptide, the specific sequence of which is shown in SEQ ID No: 2 in the sequence table.
Embodiment 3
[0021] Embodiment 3: polypeptide coupling KLH protein
[0022] Dissolve 20 mg of SMCC in 2 ml of DMF (dimethylformamide), add 0.8 ml of KLH into a 25 ml round bottom flask, and add 1×PBS (pH 7.2) to make the final protein concentration 15 mg / ml. The dissolved SMCC solution was slowly added dropwise to the 120 mg KLH protein system, and the reaction was stirred at room temperature for 1 h. Dialyze with 1L 1×PBS (pH7.4) solution at 4°C for 6 hours to remove free SMCC. Pour the dialyzed KLH protein into a 50ml centrifuge tube to obtain a volume of 20ml. Take out 417ul KLH-SMCC solution and transfer it to a 5ml centrifuge tube. 3.0 mg of polypeptide was dissolved with 0.6 ml of 1×PBS (pH 7.2) solution. The sulfhydryl group in the polypeptide was detected with E1lman reagent, and the OD value was 0.18. The polypeptide solution was added dropwise into the KLH-SMCC tube, and mixed with a vertical mixer at room temperature for 4 hours. The OD value was 0.02 when detected by Ellman...
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