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Expression and purification of murine norovirus VP1 protein virus-like particles and preparation of polyclonal antibody

A mouse and virus technology, applied in the field of molecular biology, can solve problems such as scientific research work and experimental animal production loss, and the impact of experimental results

Inactive Publication Date: 2018-05-04
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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Problems solved by technology

For mice with normal immune function, although mouse norovirus will not produce obvious pathological changes, it will also affect the experimental results and cause losses to scientific research work and experimental animal production

Method used

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  • Expression and purification of murine norovirus VP1 protein virus-like particles and preparation of polyclonal antibody
  • Expression and purification of murine norovirus VP1 protein virus-like particles and preparation of polyclonal antibody
  • Expression and purification of murine norovirus VP1 protein virus-like particles and preparation of polyclonal antibody

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Embodiment Construction

[0047] The present invention will be further described below in conjunction with the examples, and the experimental methods that do not specify specific conditions in the following examples generally follow the known methods in the art.

[0048] (1) Optimization and synthesis of MNV VP1 gene

[0049] Our laboratory isolated a mouse norovirus Nanjing1608-1 strain (GenBank: KX987840.1, the nucleotide sequence is shown in SEQ ID NO.2), and optimized the codon according to the expression preference of insect cells. The nucleotide sequence of the murine norovirus structural protein VP1 gene is shown in SEQ ID NO.1. It was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and inserted into the pUC57-simple vector through the EcoRⅤ restriction site to construct the pUC57-simple-op.VP1 recombinant plasmid, which was delivered to our laboratory. See figure 1 .

[0050] (2) Construction and identification of recombinant plasmid pFastBac Dual-op.VP1

[0051] According to the ...

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Abstract

The invention discloses expression and purification of murine norovirus VP1 protein virus-like particles and preparation of a polyclonal antibody. According to the technical scheme, structural proteinVP1 genes of the murine norovirus are subjected to codon optimization for the first time, protein is expressed by utilizing a baculovirus expression system, the expressed protein forms the virus-likeparticles, and the antibody can simulate capsid protein of live viruses. For a VLPs antigen, the new Zealand white rabbit is immunized, the polyclonal antibody capable of resisting op.VP1 is prepared, the experiment proves that the polyclonal antibody can be used for Western Blot detection and IFA analysis, and a basic material is provided for detecting the MNV carrying condition of the laboratory mouse in a laboratory. Meanwhile, the VLPs antigen and the polyclonal antibody lay a foundation for further developing an ELISA detection kit.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to expression and purification of mouse norovirus VP1 protein virus-like particles and preparation of polyclonal antibodies. Background technique [0002] Norovirus (NoV), which belongs to the family Calicivirus, is one of the important pathogens causing outbreaks of acute gastroenteritis. This virus is responsible for more than 90% of the cases of non-bacterial gastroenteritis disease affecting humans worldwide. Norovirus is a single-stranded positive-sense RNA virus with a gene length of about 7.5kb, including three open reading frames (ORFs), namely ORF1, ORF2 and ORF3. Among them, ORF1 encodes non-structural proteins including conserved RNA polymerase; ORF2 and ORF3 encode viral structural proteins, encoding capsid protein VP1 and a small basic protein VP2, respectively. Recent studies have discovered a new open reading frame ORF4, which overlaps with ORF2 but has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/866C07K16/10C07K16/06
CPCC07K14/005C07K16/06C07K16/10C12N15/86C12N2710/14043C12N2770/16022C12N2800/105C12N2800/22
Inventor 刘捷恽时锋马畅张旭亮尤金炜董敏陈莉
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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