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A bovine viral diarrhea virus-like particle and its construction method and application

A technology for bovine viral diarrhea and a construction method, which is applied to the field of bovine viral diarrhea virus-like particles and their construction, can solve the problems that the research on BVDV virus-like particles has not been carried out, and achieves the effects of low cost and high safety

Active Publication Date: 2019-06-04
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • A bovine viral diarrhea virus-like particle and its construction method and application
  • A bovine viral diarrhea virus-like particle and its construction method and application
  • A bovine viral diarrhea virus-like particle and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of recombinant transfer vector

[0051] 1. Acquisition of BVDV structural genes

[0052] There are many BVDV strains, but the structure and function are basically the same. The present invention selects the original nucleotide sequence of the C-E0-E1-E2-P7 gene region of the GenBank accession number U63479.1 strain as the research object, and optimizes it Synthesis, the nucleotide sequence was synthesized by Nanjing GenScript with a length of 2910bp, and the optimized 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 nucleotide sequence after optimized synthesis is shown in SEQ ID NO:3, and the amino acid sequence encoded by them is shown in SEQ ID NO:4.

[0054] 2. Enzyme digestion, liga...

Embodiment 2

[0060] Example 2 Construction of recombinant baculovirus

[0061] 1. Construction of recombinant baculovirus genome

[0062] Follow Invitrogen's operating instructions, 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 Spodoptera californica nuclear polyhedrosis baculovirus AcMNPV genome, Invitrogen Cat NO. 10359-016 ), the conversion conditions are as follows:

[0063] After mixing 2 μL pFastBac1-C-E0-E1-E2-P7 positive plasmid with 100 μL Escherichia coli DH10Bac competent cells, ice bath for 35 min, heat stress at 42°C for 50 s, then ice bath for 3 min, and then add 1 mL of Resistant LB medium was cultured at 37°C and 200rpm for 4-5h, and the bacterial solution was incubated for 10 hours. -1 , 10 -2 , 10 -3 Double serial dilution, take 100 μL of each dilution and spread it on a culture dish containing three-antibiotic-resistant bacteria (containing 50 μg / m...

Embodiment 3

[0066] Example 3 Rescue of recombinant baculovirus

[0067] According to the operating instructions of Invitrogen's expression system and liposome Lipfectin 2000, briefly described below. The recombinant baculovirus Bacmid plasmid extracted in Example 2 was transfected into the insect cell Spodoptera frugiperda Sf9 cell line, and the transfection process was as follows:

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

[0070] (3) the above-mentioned (1) and (2) solutions were mixed in equal volumes, and left standstill at room temperature for 20min;

[0071] (4) Wash the Sf9 cells cultured in 6-well plates (the area covering the bottom of the wells has reached 70-80%, pur...

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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 a construction method and application thereof, belonging to the technical fields of immunology and genetic engineering. Background technique [0002] Bovine viral diarrhea-mucosal disease (BVDMD) is an acute, contact infectious 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 necrosis, erosion or ulceration of the gastrointestinal mucosa. It can be transmitted both horizontally and vertically, and the virus is also present in bull semen. Pregnant cows infected with the virus are characterized by peptic ulcers. BVDV mainly infects through the respiratory tract and digestive tract, and can also infect the fetus through the placenta, giving birth to calves with virus. Most of the sick cows are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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