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Cell line for stably expressing schmallenberg virus nucleocapsid protein and application of cell line

A nucleocapsid protein, stable expression technology, applied in the field of genetic engineering and immunization, can solve the problems of long distance in the epidemic area, transmission, etc., to achieve the effect of ensuring a large amount of supply, fast growth, and simple culture conditions

Active Publication Date: 2014-12-24
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 for stably expressing schmallenberg virus nucleocapsid protein and application of cell line
  • Cell line for stably expressing schmallenberg virus nucleocapsid protein and application of cell line
  • Cell line for stably expressing schmallenberg virus nucleocapsid protein and application of cell line

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 amplification instrument, product of Applied Biosystems; 3111 water-jacketed carbon dioxide incubator, product of Thermo Electron Corporation; Nikon Eclipse Ti-U inverted fluorescence microscope, product of Nikon Company.

[0043] 2 Main reagents, consumables and cells

[0044] DMEM (Cat. No. 11995-065), Fetal Bovine Serum (Cat. No. 10099-133), Trypsin (Cat. No. 25200-056), products of Life Technologies; Gel Extraction Kit (Cat. No. D2500-01), products of OMEGA; lentiviral vector pLV - EGFP-C, auxiliary carriers pH1 and pH2, products of Beijing Yingmao Shengye Biotechnology Co., Ltd.; Qiagen Plasmid Plus Maxi Kit (Product No. 12965), products of Qiagen; EcoRI (Product No. 1040A), BamHI (Product No. 1010A) restriction end Enzymes, DNA ligase (Cat. No. 6022), One Step RNA PCR Kit (AMV) (Cat. No. RR024A), product...

Embodiment 2

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

[0074] 1 main instrument:

[0075] Bio-Rad iQ5 fluorescent quantitative PCR instrument, Bio-Rad company products.

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

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

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

[0079] Primers BHK-21-SBV-N-F(5'-TCAGATTGTCATGCCCCTTGC-3'), BHK-21-SBV-N-R(5'-TTCGGCCCCAGGTGCAAATC-3') and TaqMan probe BHK-21-SBV- for detection of SBV N gene N-Probe(5'-FAM-TTAAGGGATGCACCTGGGCCGATGGT-BHQ1-3'); Primer BHK-21-β-a...

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, product of Bio-Rad; 102 type Kodak medical X-ray film processor, product of Kodak company; EU-88 image scanner, product of Epson company; VCX605 ultrasonic breaker, Sonics&Materials company's product.

[0101] 2 Main reagents, consumables and cells

[0102]DMEM (Cat. No. 11995-065), Fetal Bovine Serum (Cat. No. 10099-133), Trypsin (Cat. No. 25200-056), products of Life Technologies; horseradish enzyme-labeled goat anti-mouse IgG (Cat. No. 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 Biotechnology Research Institute; rabbit anti-β-actin polyclonal antibody (Cat. ), product of Sigma Company; mouse ...

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Abstract

The invention provides a BHK-21 cell line for stably expressing schmallenberg virus (SBV) nucleocapsid protein. The preservation number of the BHK-21cell line is CGMCC NO. 7616, and the cell line is named BHK-21-pLV-EGFP-SBV-N. The cellular immunity fluorescence experiment result shows that the cell line can not only have specific reaction with a monoclonal antibody 2C8 of SBV, but also can recognize positive serum of SBV infected cattle. The BHK-21 cell line for stably expressing the SBV N protein can be used for detecting schmallenberg disease and has good application prospect.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and immunization, 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 arbo-borne viral disease caused by Schmallenberg virus (SBV). The vector is Culicoides, which mainly infect ruminants such as sheep, cattle and goats. , can lead to a decline in the production performance of adult livestock, and abortion, stillbirth or deformed fetus in pregnant female animals. [0003] SBV belongs to the Simbu serogroup of the family Bunyaviride and the genus Orthobunyavirus. The SBV virion is spherical, with a diameter of 80-120nm, with fibrils and capsules on the surface, and 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...

Claims

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Application Information

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Patent Type & Authority Applications(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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