Preparation method of anti-canine parvovirus protein VP2 specific IgY
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A canine parvovirus, specific technology, applied in the field of biomedicine, can solve the problems of being unsuitable for primary detection, and achieve good reactogenicity, low preparation cost, and large antibody output
Inactive Publication Date: 2014-02-12
NORTHWEST A & F UNIV
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The PCR detection method is highly sensitive, but requires special equipment, and is not suitable for the needs of grassroots detection
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[0026] A preparation method of anti-canine parvovirus VP2 protein IgY, said method comprising the following steps:
[0027] (1) Cloning and sequence analysis of canine parvovirus VP2 gene. According to the relevant CPV-VP2 gene sequence reported by GenBank, a pair of primers were designed using primer prime5.0 software, and the CPV-VP2 gene (1755bp) was amplified by PCR, connected to the pMD18-T vector, transformed into DH5α competent cells, and passed blue-white spot After screening, plasmids were extracted, analyzed by enzyme digestion, and positive plasmids were sequenced.
[0028](2) Expression and purification of VP2 protein in Escherichia coli. The pMD18-T-VP2 and pET-32a vectors were digested with BamHI and XhoI, the target fragments were connected, and the pET-32a-VP2 expression vector was constructed, and transformed into Bal21 (DE3) pLysS competent bacteria. After enzyme digestion and PCR identification, IPTG was optimized The concentration and time of induction we...
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[0031] This example intends to clone the CPV-VP2 structural protein, construct the prokaryotic expression vector pET-32a-VP2, express and purify the VP2 protein, and use it as an immunogen to immunize laying hens to produce anti-VP2 recombinant protein-specific IgY antibodies .
[0032] 1. Primer design and synthesis
[0033] According to the sequence of canine parvovirus reference strain (FJ011098) registered in GenBank, a pair of primers were designed using the software Primer Prime5.0 to amplify the full-length DNA sequence of VP2 gene (1755bp). Upstream primer P: 5, -ATGGATCCCCAATGAGTGATGGAGCAG-3, (underlined is the BamH I restriction site), downstream primer R: 5, -CCGCTCGAGATATAATTTTCTAGGTGCTAGTTG-3, (the underline is the Xho I restriction site). The primers were synthesized by Shanghai Sangong Company, diluted with double distilled water to 10 pmol / μL, and stored at -20°C.
[0034] 2. PCR amplification of VP2 gene
[0035] Template preparation: Take 50 μL of virus su...
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Abstract
The invention discloses a preparation method of an anti-canine parvovirus protein VP2 specific IgY, which comprises the following steps: (1) designing a pair of primers according to CPV-VP2 gene sequence, carrying out PCR (polymerase chain reaction) amplification on the CPV-VP2 gene, connecting to a pMD18-T vector, transforming DH5alpha competent cell, carrying out blue-white screening, extracting the plasmid, carrying out digest enzyme digestion ion analysis, carrying out positive plasmid sequencing, and carrying out comparative analysis on the sequencing result; (2) expression and purification of VP2 protein in Escherichia coli: carrying out BamH I and Xho I double-enzyme digestion on the pMD18-T-VP2 and pET-32a,connecting to the target segment, constructing the pET-32a-VP2 expression vector, transforming Bal21(DE3)pLysS competent bacteria, carrying out enzyme digestion and PCR identification, optimizing the IPTG (isopropyl-beta-D-thiogalactopyranoside) induction concentration and time, carrying out mass induction expression, and purifying the recombinant protein by using a Ni<+> affinity column; and (3) preparation of anti-VP2-IgY antibody: immunizing a laying hen by using the purified VP2 protein, extracting the specific IgY antibody by using PEG 6000, and carrying out SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. The anti-VP2-IgY antibody extracted by the method can be well combined with the VP2 protein to carry out non-cross reaction on the degradation segment.
Description
technical field [0001] The invention belongs to the field of biomedicine and relates to the preparation and application of a specific IgY, in particular to a method for preparing an anti-acid orange II egg yolk antibody. Background technique [0002] Canine parvovirus (Canine parvovirus, CPV) can cause hemorrhagic enteritis and myocarditis in dogs, and 6-month-old animals are more common, accounting for 65.7% of the annual incidence. There is little difference in popular seasons, with slightly more in spring and autumn (Liu Dafei et al. 2009). In China, canine parvovirus disease was first reported in 1983, which seriously endangered the dog industry in my country and caused serious economic losses. [0003] CPV is a single-stranded DNA virus, containing 5323 nucleotides, including two ORFs (Open reading frame, ORF), the 3' end ORF encodes non-structural proteins NS1 and NS2, and the 5' end ORF encodes structural proteins VP1 and VP2 (Zhao et al. 2011). The viral empty cap...
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