Kit for quantitatively detecting NT-proBNP (N-terminal prohormone Brain Natriuretic Peptide) and preparation method of kit
A quantitative detection and kit technology, applied in the fields of peptide chemistry and immunology, can solve the problems of poor sensitivity and cumbersome detection operation, and achieve the effect of high sensitivity, good repeatability and rapid detection
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Embodiment 1
[0063] The preparation method of NT-proBNP natural protein is as follows:
[0064] 1. Design primers according to the DNA sequence of human NT-proBNP provided in Genbank. The 5'ends of the primers are introduced into NdeI+XhoI restriction sites respectively, and the target gene of NT-proBNP is obtained by PCR amplification. The vector pET-28a and The NT-proBNP gene fragment purified by agarose gel was treated with NdeI+XhoI for double enzyme digestion, and T 4 DNA ligase ligates the purified digested product to obtain recombinant plasmid pET-28a-NT-proBNP, and the ligated product is transformed into E. coli DH5α. The clones are selected on the LB plate containing ampicillin, and the plasmid is prepared in a small amount by double enzyme Digestion / PCR identification screened out positive clones, and the sequencing results showed that the recombinant NT-proBNP fragment was completely consistent with the designed sequence.
[0065] 2. After the NT-proBNP plasmid is verified by sequenc...
Embodiment 2
[0069] The preparation method of NT-proBNP peptide is as follows:
[0070] The following 4 groups of peptides were synthesized by Shanghai Jier Biochemical and Biological Company; the purpose of amino acid changes is to change hydrophobic amino acids into hydrophilic amino acids and increase the hydrophilicity of the protein. At the same time, the target protein of the sample is tested in subsequent experiments to screen out the most Suitable antibodies.
[0071] The epitope peptide of the present invention is peptide 1, numbered as SEQ ID NO. 1: GESDPLGSPGSASDLETSGL.
[0072] Peptide 2, numbered as SEQ ID NO. 2: LQVKQTSLEPLQESPRPTG.
[0073] Peptide 3, numbered as SEQ ID NO. 3: ESPRPTGVWKSDEVATEGIRG.
[0074] Peptide 4, numbered as SEQ ID NO. 4: RAPESPKMVQGSGCFGRKMDRI.
Embodiment 3
[0076] Prepare NT-proBNP natural protein specific antibody, NT-proBNP natural protein specific antibody as capture antibody, and prepare NT-proBNP peptide specific antibody, NT-proBNP peptide specific antibody as detection antibody, and capture antibody And the detection antibody for purification, the method is as follows:
[0077] Use NT-proBNP natural protein and 4 groups of peptides to immunize 2 healthy female BALB / c mice at the age of 8 weeks, weighing about 18g, and after adaptive breeding for 1 week, collect negative blood as a control; use medium-range immunization Protocol (0.3ml / mouse, 2 weeks / time), for the first immunization (50μg / mouse), stir and emulsify the immunogen with an equal volume of Freund’s complete adjuvant, and then subcutaneously inject multiple points on the back, then press the immunogen and equal volume Freund's incomplete adjuvant is stirred and emulsified for routine immunization; for 3 immunizations, generally 50μg antigen and TiterMax are mixed a...
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