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Preparation method of GyV7 ring virus VP3 protein

A circular virus and protein technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve problems such as low efficiency and cumbersome cloning process, achieve rapid cloning and expression, and simplify the cloning process

Inactive Publication Date: 2017-01-25
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But sometimes the cloning process is cumbersome and inefficient due to the inability to find a suitable restriction site

Method used

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  • Preparation method of GyV7 ring virus VP3 protein
  • Preparation method of GyV7 ring virus VP3 protein
  • Preparation method of GyV7 ring virus VP3 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1) Design and amplify primers containing pGEX-6p-1 linearized vector and GyV7 virus VP3 gene fragment:

[0032] The upstream primers for the amplified pGEX-6p-1 linearized vector are located at positions 1022-1047 of the pGEX-6p-1 plasmid; and there are additional TAA bases at the 5'end; the downstream primers for the amplified pGEX-6p-1 linearized vector are located at pGEX -6p-1 plasmid at positions 916-941; and has an extra CAT base at the 5'end. The upstream primers for the amplification of the GyV7 virus VP3 gene include the VP3 gene start code ATG and the following 14 bases, and the 5'end has 16 reverse complementary bases with the downstream primers of the pGEX-6p-1 linearized vector; The downstream primer of VP3 gene of augmented GyV7 virus includes the stop code TAA of VP3 gene and its first 14 bases, and has 16 reverse complementary bases at the 5'end of the upstream primer of pGEX-6p-1 linearized vector. The specific primer sequences are shown in Table 1, synth...

Embodiment 2

[0040] GyV7 virus VP3 protein induced expression and purification: The obtained positive clone containing GyV7 virus VP3 gene (named pGEX-VP3) was transformed into BL21 bacteria, and the bacteria were collected after induction by IPTG (0.1mmol / ml), and then subjected to ultrasound (40 Hz). )broken. Centrifuge the ultrasonically broken samples and separate the supernatant and the pellet for SDS-PAGE (5% concentrated gel, 10% separating gel) and Western blot analysis (using anti-mouse GST monoclonal antibody as primary antibody, goat anti-mouse HRP labeled IgG is the secondary antibody) to identify the expression.

[0041] The specific steps are as follows:

[0042] 1) GyV7 virus VP3 protein induced expression:

[0043] After the obtained positive clone containing the GyV7 virus VP3 gene (named pGEX-VP3) was transformed into BL21 bacteria, it was transferred to 3ml of LB liquid medium containing Amp (100μg / mL) and cultured overnight at 37°C with shaking at 250rpm. The next day, it w...

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Abstract

The invention provides a preparation method of GyV7 ring virus VP3 protein. In the method, based on a recombinase Exnase TM II, a linearized pGEX-6p-1 carrier and a PCR product of GyV7 virus VP3 gene segment are directly treated by a recombinant cloning technology without an enzyme-cut link up reaction and then transformed into escherichia coli to realize expression of the VP3 protein, wherein the nucleotide sequences of the upstream and downstream primers applied to the PCR amplification of GyV7 virus VP3 gene are shown by SEQ ID No.3 and SEQ ID No.4 respectively. The method is easy to operate and quick and efficient; the obtained protein expressed and purified by the GyV7 virus VP3 can directly supply VP3 protein as a GyV7 diagnosis antigen; the VP3 protein serves as an immunogen to obtain an anti-VP3 polyclonal antibody; an effective diagnosis reagent is provided for developing GyV7 epidemiological investigation; and the VP3 protein is of great significance to further study of the biological functions of VP3.

Description

Technical field [0001] The invention relates to a method for preparing GyV7 circular virus VP3 protein. Background technique [0002] Chicken Infectious Anemia Virus (CAV) has always been regarded as the only member of Gyrovirus in the Circoviridae family. Until 2011, Rijsewijk et al. (Rijsewijk FA, Dos Santos HF, Teixeira TF, Cibulski SP, Varela AP, Dezen D, etal. Discovery of a genome of a distant relative of chicken anemia virusreveals a new member of the genus Gyrovirus.Archives of virology. 2011Jun; 156(6):1097-100), a novel circovirus sequence was detected from the serum samples of diseased chickens, the second member of the circovirus genus, named AGV2. In the same year, Sauvage et al. (Sauvage V, Cheval J, Foulongne V, Gouilh MA, Pariente K, Manuguerra JC, et al. Identification of the first human gyrovirus, a virus related to chicken anemia virus. Journal of virology. 2011Aug; 85(15): 7948-50) The first human circovirus HGyV sequence highly homologous to AGV2 was detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/70C12N15/11
CPCC07K14/005C12N15/70C12N2750/10022C12N2800/101
Inventor 叶建强申秋平邵红霞秦爱建田晓彦万志敏
Owner YANGZHOU UNIV
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