Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide
A monoclonal antibody and B cell technology, applied in the field of immunoanalysis medicine, can solve the problems of low product yield, complicated preparation process, and high cost, and achieve high titer, high purity, and good specificity
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Embodiment 1
[0031] The preparation of embodiment 1 PCT recombinant protein
[0032] On the basis of obtaining the PCT coding gene from GENBANK, carry out codon bias modification, chemically synthesize the coding gene fragment, clone into the prokaryotic expression vector and induce protein expression after identification by DNA sequencing. The protein can be used as an immunogen and screening agent for antibody preparation, and can also be used as a calibrator for the establishment of PCT quantitative detection in subsequent experiments, specifically:
[0033] A PCT recombinant protein gene clone:
[0034] Obtain the gene sequence of human PCT protein from GENBANK (accession number is NM_004102), submit it to Graphical codon usage analyzer (http: / / guca.schoedl.del) to analyze its codon bias; specifically, human PCT The synonymous substitution of gene codons with a usage rate of <10% in Escherichia coli is replaced by codons preferred by Escherichia coli to make it easier to express in ...
Embodiment 2
[0063] Example 2 Preparation of B cell epitope peptides of human PCT
[0064] The sequence of the chemically synthesized B cell epitope peptide is from the results of bioinformatics analysis, comprehensively analyzing the secondary structure, antigenicity, hydrophilicity, accessibility and flexibility of the PCT protein, and analyzing and scoring each segment, The region with high score was selected as the B cell epitope region. Specifically, Chou & Fasman was used to predict β-turn angle, Emini method to predict antigen surface accessibility, Karplus & Schulz method to predict protein flexibility, Kolaskar & Tongaonkar protein antigenicity analysis, Parker method protein hydrophobicity analysis and Bepipred linear epitope prediction technology. Parameters to obtain the B cell epitope peptide of human PCT;
[0065]The screening B cell epitope peptide, wherein the chemically synthesized epitope peptide is: N-- cmlgtytqdfnkfh—C (SEQ ID NO: 3), the B cell epitope peptide chemi...
Embodiment 3
[0068] Example 3 Preparation and Identification of Monoclonal Antibody
[0069] Immunization of Balb / c mice with a B cell epitope peptide derivative
[0070] The B cell epitope peptide antigen obtained in Example 2 was taken out from the -80°C refrigerator as the antigen, dissolved and then filtered.
[0071] Female Balb / c mice aged 6 weeks and weighing about 20 g were selected for immunization. Antigen emulsification adopts double syringe mutual pushing method. For the first immunization, the B cell epitope peptide antigen shown in SEQ ID NO: 4 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain an antigen mixture, which was added intradermally in an amount of 100 μg to each mouse intraperitoneal injection. The second and third immunizations were carried out on the 14th and 28th days respectively. The adjuvant was changed to incomplete Freund's adjuvant. The amount of antigen, injection volume and route remained unchanged. After the thir...
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