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Cell line stably expressing Schmallenberg virus nucleocapsid protein and its application

A nucleocapsid protein, stable expression technology, applied in the field of genetic engineering and immunization, can solve the problems of introduction and long distance in the epidemic area, and achieve the effects of fast growth, large supply, and convenient observation and analysis

Active Publication Date: 2016-08-17
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the spatial distance between our country and the European epidemic area is far away, it is difficult for the disease to spread to our country through the flight of poisonous Culicoides under natural conditions. Increasingly frequent, the disease has the potential risk of being introduced into my country

Method used

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  • Cell line stably expressing Schmallenberg virus nucleocapsid protein and its application
  • Cell line stably expressing Schmallenberg virus nucleocapsid protein and its application
  • Cell line stably expressing Schmallenberg virus nucleocapsid protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of BHK-21 cell line stably expressing Schmallenberg virus nucleocapsid protein

[0041] 1 main instrument

[0042] ABI Veriti 96-well PCR Amplifier, Product of Applied Biosystems; Model 3111 Water Jacketed Carbon Dioxide Incubator, Product of Thermo Electron Corporation; Nikon Eclipse Ti-U Inverted Fluorescence Microscope, Product of Nikon Company.

[0043] 2Main reagents, consumables and cells

[0044] DMEM (product number 11995-065), fetal bovine serum (product number 10099-133), trypsin (product number 25200-056), product of Life Technologies; Gel Extraction Kit (product number D2500-01), product of OMEGA; lentiviral vector pLV -EGFP-C, auxiliary carrier pH1 and pH2, products of Beijing Yingmao Shengye Biotechnology Co., Ltd.; Qiagen Plasmid Plus Maxi Kit (product number 12965), products of Qiagen company; EcoRI (product number 1040A), BamHI (product number 1010A) restriction endonucleases , DNA Ligase (Cat. No. 6022), One Step RNA PCR Kit (AMV) (Cat....

Embodiment 2

[0073] Example 2 Fluorescence quantitative RT-PCR identification of BHK-21 cell line stably expressing Schmallenberg virus nucleocapsid protein

[0074] 1 Main instruments:

[0075] Bio-Rad iQ5 fluorescent quantitative PCR instrument, a product of Bio-Rad.

[0076] 2 Main reagents, cells and primers, probes:

[0077] Mini Kit (article number 74104), a product of Qiagen; AgPath-ID TM One-Step RT-PCR Kit (Cat. No. AM1005), a product of Life Technologies.

[0078] BHK-21 cells (BHK-21-EGFP-SBV-N) stably expressing SBV N protein, BHK-21 cells (BHK-21-EGFP) and BHK-21 cells stably expressing enhanced green fluorescent protein (EGFP): All are prepared and preserved by the Animal Quarantine Laboratory of the Chinese Academy of Inspection and Quarantine.

[0079] The primers BHK-21-SBV-NF (5'-TCAGATTGTCATGCCCCTTGC-3'), BHK-21-SBV-NR (5'-TTCGGCCCCAGGTGCAAATC-3') and TaqMan probe BHK-21-SBV- for detecting SBV N gene N-Probe (5'-FAM-TTAAGGGATGCACCTGGGCCGATGGT-BHQ1-3'); primer BHK-21-β-actin-F ...

Embodiment 3

[0098] Example 3 Western Blot identification of BHK-21 cell line stably expressing Schmallenberg virus nucleocapsid protein

[0099] 1 main instrument

[0100] PowerPac1000 protein electrophoresis instrument and its transfer system, Bio-Rad product; 102 Kodak medical X-ray developing machine, Kodak product; EU-88 image scanner, Epson product; VCX605 ultrasonic disintegrator, Sonics&Materials company's product.

[0101] 2Main reagents, consumables and cells

[0102] DMEM (product number 11995-065), fetal bovine serum (product number 10099-133), trypsin (product number 25200-056), product of Life Technologies; horseradish enzyme-labeled goat anti-mouse IgG (product number ZB-2305), horseradish Enzyme-labeled goat anti-rabbit IgG (Cat. No. ZB-2301), product of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; RIPA cell lysate (Cat. No. P0013B), product of Biyuntian Institute of Biotechnology; Rabbit anti-β-actin polyclonal antibody (Cat. No. A2066) ), a product of Sigma; mouse anti-en...

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Abstract

The present invention provides a BHK-21 cell line stably expressing Schmallenberg virus (SBV) nucleocapsid protein, its preservation number is CGMCC No.7616, named BHK-21-pLV-EGFP-SBV-N . The results of cell immunofluorescence test showed that the cell line not only reacted specifically with SBV monoclonal antibody 2C8, but also recognized the positive serum of SBV-infected cattle. The BHK‑21 cell line stably expressing SBV N protein can be used for the detection of Schmallenberg disease and has a good application prospect.

Description

Technical field [0001] The present invention relates to the field of genetic engineering and immune technology, in particular to a BHK-21 cell line stably expressing Schmallenberg virus nucleocapsid protein and a preparation method thereof. Background technique [0002] Schmallenberg disease (Schmallenberg disease) is an arbovirus disease caused by Schmallenbergvirus (SBV). The vector is Culicoides. It mainly infects ruminants such as sheep, cattle and goats. It can lead to decreased performance of adult livestock, miscarriage, stillbirth or malformed fetus in pregnant mothers. [0003] SBV belongs to the Bunyaviride family (Bunyaviride) Orthobunyavirus (Orthobunyavirus) Simbu serogroup (Simbu serogroup). SBV virus particles are spherical in shape, 80-120nm in diameter, with fibrils and envelopes on the surface. The genome is a single-stranded negative-strand RNA, which can be divided into three gene segments, L, M, and S. L encodes RNA-dependent RNA polymerase, M encodes two sur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12P21/02G01N33/569C12R1/91
Inventor 张永宁林祥梅吴绍强吕继洲冯春燕邓俊花袁向芬
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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