Preparation method of virus-like particles of muscovy duck parvovirus

A Muscovy duck parvovirus, virus-like technology, applied in the direction of botany equipment and methods, biochemical equipment and methods, viruses, etc.

Inactive Publication Date: 2017-01-25
朱善元 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Preparation method of virus-like particles of muscovy duck parvovirus
  • Preparation method of virus-like particles of muscovy duck parvovirus
  • Preparation method of virus-like particles of muscovy duck parvovirus

Examples

Experimental program
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Embodiment 1

[0027] Example 1 Construction and Identification of Transfer Vector pFastBac1-VP3

[0028] According to the published sequence of Muscovy duck parvovirus VP3 gene (GeneBank accession number: FJ480824), design a pair of primers, VP3-F / R,

[0029] VP3-F: 5'-TAAGCGGCCGCCATGGCAGAGGGAGGAAGC-'3 (SEQ ID No.1)

[0030] VP3-R: 5'-GGACTCGAGTTACAGATTCTGAGTCAAAT-'3 (SEQ ID No.2)

[0031] Using the extracted Muscovy duck parvovirus-FJ strain DNA as a template, the target fragment VP3 gene was amplified by PCR. After recovery, the target fragment was digested, ligated with the vector pFastBac1 that had been digested by the same enzyme, and identified by enzyme digestion and sequencing to obtain the transfer vector pFastBac1-VP3.

Embodiment 2

[0032] Transposition of embodiment 2pFastBac1-VP3 recombinant plasmid

[0033] The pFastBac1-VP3 recombinant plasmid was transformed into DH10Bac competent cells, plated and cultured until blue and white spots appeared.

Embodiment 3

[0034] Obtaining and identification of embodiment 3 recombinant Bacmid DNA

[0035] Pick the white single colony in the blue-white spot, extract the recombinant Bacmid DNA by alkaline cleavage method, and carry out PCR identification on it with VP3 and M13 (M13 primer is a known sequence, see Invitrogen company Bac-to-Bac manual) primers respectively, the result Such as figure 1 As shown, specific amplified bands can be seen, and the size is consistent with the expectation, indicating that the homologous recombination of the target gene is successful and the recombinant bacmid obtained is correct.

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Abstract

The invention discloses a preparation method of virus-like particles of a muscovy duck parvovirus. The method comprises the following steps: cloning a gene VP3 of the muscovy duck parvovirus into a baculovirus transfer vector to obtain a recombinant transfer vector pFastBacl-VP3; converting a recombinant plasmid of the pFastBacl-VP3 into DH10Bac competent cells, and performing coated plate cultivation till blue-white spots appear; picking white single colonies, extracting a recombinant Bacmid DNA by an alkaline cracking method, and performing transfection on the recombinant Bacmid DNA to enable an insect cell Sf9 to grow to prepare a rcombinant bculovirus, wherein SDS-PAGE, Western blot and IFA results show that a recombinant protein VP3 is successfully expressed in the insect cell; infecting the insect cell with P3 replacing the virus, collecting cell cracking supernate, and performing sucrose density gradient centrifugal purification to obtain spherical virus-like particles with the diameter of about 20 to 25 nm, wherein the spherical virus-like particles are highly similar to natural DPVs (Duck Parvovirus).

Description

technical field [0001] The invention relates to the preparation of Muscovy duck parvovirus virus-like particles. Background technique [0002] Muscovy duck parvovirus disease is one of the diseases that seriously endanger the preservation of duck breeds. The disease is an acute, septic infectious disease caused by Muscovy duck parvovirus (DPV), which is characterized by high infectivity and mortality. The disease mainly occurs in young muscovy ducks within 3 weeks of age (commonly known as three-week disease of young muscovy ducks), and the morbidity and mortality can reach more than 40% to 50%. The development of duck industry in China. [0003] Muscovy duck parvovirus particles are spherical or hexagonal, non-encapsulated, icosahedral symmetry, 20-40nm in diameter, nucleic acid is single-stranded DNA, and there are two virion forms: hollow and solid. The genome of Muscovy duck parvovirus is a single-stranded linear DNA with a total length of 5136nt. MDPV contains two m...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/866
CPCC12N7/00C12N15/86C12N2710/14043C12N2750/14023C12N2800/105
Inventor 朱善元王安平吴萌王永娟左伟勇吴双郭长明秦枫洪伟鸣
Owner 朱善元
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