Porcine parvovirus VP2 affinity peptide and coding nucleotide thereof
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A parvovirus and nucleotide technology, applied in the biological field, to achieve the effects of strong penetration, easy penetration into tissues, and short half-life
Inactive Publication Date: 2013-06-12
NORTHEAST AGRICULTURAL UNIVERSITY
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[0003] At present, porcine parvovirus infection is very serious in my country, and the positive rate can reach more than 90%, which has caused huge economic losses to the pig industry
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Embodiment 1
[0032] Construction of Recombinant Expression Plasmid pET32a-VP2
[0033] (1) Amplification of VP2 sequence
[0034] (a) Design primers VP2-3 and VP2-4 according to the codons favored by bacteria
[0035] VP2-3: 5'-GG GGATCC ATGAGTGAAAATGTGGAA-3' (the dashed line indicates that the introduced enzyme cutting site is BamHI)
[0036] VP2-4: 5'-CCCC GTC GAC TTAGTATAATTTTCTTGG-3' (the dashed line indicates that the introduced enzyme cutting site is SalI)
[0037] (b) Perform conventional chain polymerase reaction to amplify the VP2 gene
[0038] Use VP2-3 and VP2-4 as primers, VP2-T-6 as template, and perform PCR with ExTaq polymerase. PCR reaction conditions for VP2: 50 μL of the PCR reaction system contained in 50 μL system, including 5 μL of 10×EasyTaq Buffer , 4 μL dNTP, 1 μL VP2-3, 1 μL VP2-4, 0.5 μL VP2-T-6, 0.5 μL ExTaq polymerase, add sterile water to make up to 50 μL. The reaction program of the mixed solution in the PCR detection is: pre-denaturation at 95°C for 5...
Embodiment 2
[0041] Example 2 PCR and Enzyme Digestion Identification of Recombinant Expression Plasmid pET32a-VP2
[0042] Select positive clones, extract plasmid pET32a-VP2, use VP2-3 and VP2-4 as primers for PCR identification, and perform double enzyme digestion with BamH I and Sal I, and detect the product by 1% agarose gel electrophoresis, and its position and prediction unanimous. The result is as figure 2 , indicating that the VP2 gene was successfully connected into the pET32a vector.
Embodiment 3
[0043] SDS-PAGE analysis of embodiment three induced expression products
[0044] The correctly identified recombinant plasmid pET32a-VP2 was transformed into E.coli Rosetta (DE3) competent cells, and IPTG was added to make the final concentration 0.6mmol / L, and cultured with shaking at 37°C. The test samples were processed and analyzed by SDS-PAGE electrophoresis. The result is as Figure 4 .
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Abstract
The invention relates to porcine parvovirus VP2 affinity peptide and coding nucleotide of the porcine parvovirus VP2 affinity peptide. An amino acid sequence of porcine parvovirus VP2 affinity peptide A is shown in a sequence table SEQ ID No.1, and a nucleotide sequence of the porcine parvovirus VP2 affinity peptide A is shown in a sequence table SEQ ID No.2. An amino acid sequence of porcine parvovirus VP2 affinity peptide B is shown in a sequence table SEQ ID No.3, and a nucleotide sequence of the porcine parvovirus VP2 affinity peptide B is shown in SEQ ID No.4. The invention further relates to nucleotide sequences of a pair of primers VP2-3 and VP2-4. Due to the fact that the molecular weight of polypeptide in the porcine parvovirus VP2 affinity peptide is small, the porcine parvovirus VP2 affinity peptide can penetrate into tissue easily, the porcine parvovirus VP2 affinity peptide is stronger than proteins in infiltration capacity, the half-life period of the porcine parvovirus VP2 affinity peptide is short, accumulation in the tissue is little, the structure is stable and production cost is low.
Description
technical field [0001] The invention relates to a porcine parvovirus VP2 affinity peptide and its coded nucleotide, belonging to the field of biotechnology. Background technique [0002] Phage surface display technology (phage display random peptide library) is a new molecular biology technology developed in the 1980s that uses phage to express foreign genes. The phage vectors selected for phage display technology are filamentous phages, including f1, fd and M13 phages, among which M13 phages are most commonly used. The phage of this type of single-stranded circular DNA virus particle encodes 11 proteins, pIII, pVI, pVII, pVIII, and pIX are its capsid structural proteins, and the pVIII protein is the main capsid protein, which constitutes the tubular structure of the phage and is helical. Symmetrically arranged, pIII and pVII proteins are located at the tail end of the phage shell, which is the structure for the phage to adsorb the host bacteria. At present, most studies ...
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