Rapid quantitative detection card for canine distemper virus antibody and using method
A canine distemper virus, quantitative detection technology, applied in the field of detection cards, can solve the problems of narrow detection range, only qualitative, low sensitivity, etc., to achieve the effect of improving sensitivity, small intra-batch variation, and high sensitivity
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Embodiment 1
[0033] Embodiment 1, eukaryotic expression of canine distemper virus structural protein H protein
[0034]Step 1. Primer design and PCR amplification of the target gene
[0035] Design specific upstream primer SP1: 5'-TCGAATTCATGAAAAAACTATTTGTG-3' according to the nucleotide sequence of canine distemper virus H gene published in GenBank database; downstream primer SP2: 5'-TTGCGGCCGCTTACATCACATGGCGT-3', the italic black part is the enzyme cut site. Pichia P1: 5'-GACTGGTTCCAATTGACAAGC-3'; P2: 5'-GCAAATGGCATTCTGACATCC-3'. The synthetic hemagglutinin gene (Attachment protein gene, H) genome clone was used as a template, primers SP1 and SP2 were used for PCR amplification reaction, and the final product was checked by 0.8% agarose gel electrophoresis.
[0036] Step 2. Construction and identification of cloning vector TS
[0037] After the target fragment was recovered by the gel recovery kit, it was ligated with the pBS-T vector at 16°C and transformed into Escherichia coli comp...
Embodiment 2
[0048] Example 2: Canine Distemper Virus Structural Protein H Protein Using Specific Synthetic Peptides to Prepare Antigen
[0049] A method for preparing the above-mentioned canine distemper virus structural protein H protein, comprising the following steps:
[0050] Step 1. Sequence analysis of canine distemper virus structural protein H protein: use bioinformatics to predict MHC class I molecules: verify through relevant websites or use computer software to analyze the sequence of CDV-H protein to understand its hydrophilicity, hydrophobicity, Domain accessibility, sequence variability, α-helix, β-turn, antigenicity and other parameters, and then comprehensive analysis and homology modeling methods to predict its tertiary structure, from which the antigenic reactive epitope and amino acid residues are predicted , according to the degree of difficulty of the peptide synthesizer for comprehensive analysis design. Each polypeptide chain contains at least one predicted antigen...
Embodiment 3
[0052] Embodiment three, the detection card of preparation canine distemper virus antibody
[0053] Preparation of canine distemper virus antibody detection standard curve: prepare 6 copies of calibration solution containing canine distemper virus antibody (including canine distemper virus antibody standard), the concentrations are 0, 1 / 1024, 1 / 256, 1 / 64, 1 / 16, 1 / 4 (double dilution of canine distemper virus antibody standard). Add the above-mentioned calibration solutions of different concentrations into the sample holes of the assembled test card, and after 15 minutes of chromatography, the test is carried out by a tomographic scanner, and the test results obtained 6 times are processed by the client, and the client calculates The fluorescence signal intensity values of the detection line and quality control line corresponding to the standard product, and perform linear regression based on this data to make a standard curve for canine distemper virus antibody. The standard ...
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