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Bovine viral diarrhea virus-like particle and construction method and application thereof

A technology of bovine viral diarrhea and its construction method, which is applied in the field of bovine viral diarrhea virus-like particles and its construction, can solve the problems that the research on BVDV virus-like particles has not yet been carried out, and achieve the effect of low cost and high safety

Active Publication Date: 2018-12-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, research on BVDV virus-like particles (BVDVLPs) has not been carried out

Method used

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  • Bovine viral diarrhea virus-like particle and construction method and application thereof
  • Bovine viral diarrhea virus-like particle and construction method and application thereof
  • Bovine viral diarrhea virus-like particle and construction method and application thereof

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Experimental program
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Embodiment 1

[0050] The construction of embodiment 1 recombinant transfer vector

[0051] 1. Acquisition of BVDV structural gene

[0052] There are many BVDV strains, but the structure and function are basically the same. In the present invention, the original nucleotide sequence of the C-E0-E1-E2-P7 gene region of the strain with GenBank accession number U63479.1 is selected as the research object and optimized. Synthesized, the nucleotide sequence was synthesized by Nanjing GenScript with a length of 2910bp, and the optimized and synthesized target sequence was connected to the PUC57 vector (purchased from Thermo Fisher Scientific), named PUC57-C-E0-E1-E2- P7.

[0053] The original nucleotide sequence of the BVDV C-E0-E1-E2-P7 gene region is shown in SEQ ID NO:1, and the amino acid sequence encoded by them is shown in SEQ ID NO:2. The optimized and synthesized nucleotide sequence is shown in SEQ ID NO:3, and the amino acid sequence encoded by them is shown in SEQ ID NO:4.

[0054] 2. ...

Embodiment 2

[0060] The construction of embodiment 2 recombinant baculovirus

[0061] 1. Construction of recombinant baculovirus genome

[0062] In accordance with the operating instructions of Invitrogen, briefly described below. The pFastBac1-C-E0-E1-E2-P7 positive plasmid obtained in Example 1 was transformed into Escherichia coli DH10Bac competent cells (containing the Autographa californica nuclear polyhedrosis baculovirus AcMNPV genome, Invitrogen Cat NO.10359-016 ), the conversion conditions are as follows:

[0063] Mix 2 μL of pFastBac1-C-E0-E1-E2-P7 positive plasmid with 100 μL of Escherichia coli DH10Bac competent cells, and then bathe in ice for 35 minutes. Resistant LB medium, after culturing at 37°C and 200rpm for 4-5 hours, the bacterial solution was subjected to 10 -1 、10 -2 、10 -3 Doubling serial dilutions were taken, and 100 μL of each dilution was spread on a culture dish containing three-antibody-resistant bacteria (containing 50 μg / mL kanamycin, 7 μg / mL gentamicin,...

Embodiment 3

[0066] Embodiment 3 Rescue of recombinant baculovirus

[0067] In accordance with the Invitrogen company's expression system and liposome Lipfectin 2000 operating instructions, briefly described as follows. The recombinant baculovirus Bacmid plasmid that embodiment 2 extracts is transfected insect cell Spodoptera frugiperda (Spodopterafrugiperda) Sf9 cell line, and transfection process is as follows:

[0068] (1) Add 4 μg of the recombinant baculovirus Bacmid plasmid to 250 μL of Grace medium without serum and double antibody, mix well, and let stand at room temperature for 5 minutes;

[0069] (2) Add 6 μL liposome Lipfectin 2000 to 250 μL Grace culture medium without serum and double antibody, mix well, and let stand at room temperature for 5 minutes;

[0070] (3) Mix equal volumes of the above (1) and (2) solutions, and let stand at room temperature for 20 minutes;

[0071] (4) Sf9 cells cultured in a 6-well plate (covering 70-80% of the bottom area of ​​the well, purchase...

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Abstract

The invention relates to a bovine viral diarrhea virus-like particle and a construction method and application thereof. According to the invention, the bovine viral diarrhea virus C-E0-E1-E2-P7 gene is firstly connected with a baculovirus expression vector, and then a baculovirus expression system is used to safely and efficiently produce bovine viral diarrhea virus-like particles in insect cell bioreactors. The prepared bovine viral diarrhea virus-like particles are similar in morphology to the real virus particles. Compared with the inactivated virus, the virus-like particle immunization caneffectively induce the cellular immune response without the risk of virulence recovery of the attenuated vaccine. Compared with other vaccines, the virus-like particles can more realistically displaythe viral antigen conformation, are easy to recognize by the immune system, and efficiently induce the body to produce neutralizing antibodies, thus filling the blank of such vaccines in China. The preparation method of the invention is suitable for industrial production, and has the advantages of low cost, high safety and the like.

Description

technical field [0001] The invention relates to a bovine viral diarrhea virus-like particle and its construction method and application, belonging to the technical fields of immunology and genetic engineering. Background technique [0002] Bovine viral diarrhea-mucosal disease (BVDMD) is an acute and contagious disease caused by bovine viral diarrhea viruses (BVDV). In addition to infecting cattle, BVDV can also infect pigs, deer, sheep, camels and other wild animals, with a wide range of hosts. The disease is mainly characterized by diarrhea and gastrointestinal mucosal necrosis, erosion or ulceration. It can be transmitted both horizontally and vertically. The virus is also present in bull semen. Pregnant cows infected with the virus are characterized by peptic ulcers. BVDV mainly infects the respiratory tract and digestive tract, and can also infect the fetus through the placenta, and deliver infected calves. Most sick cattle are persistently infected, which can easily...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/40C12N15/866A61K39/12A61P31/14
CPCA61K39/12A61P31/14C07K14/005C12N15/86C12N2710/14143C12N2770/24323C12N2770/24334
Inventor 郑文文郑学星赵丽宋艳艳薛付忠温红玲
Owner SHANDONG UNIV
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