Antibody, test strip and kit against bovine nodular skin disease virus

A technology for nodular and skin diseases, applied in the field of immunity, can solve the problems of expensive, complicated and time-consuming separation and culture identification methods

Active Publication Date: 2022-06-07
北京弘进久安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the cumbersome and time-consuming operation of the separation and culture identification method, the PCR method, FQ-PCR and other above-mentioned methods require expensive instruments and reagents, and the detection cost is high, so it is not suitable for laboratory and on-site detection applications in grassroots units

Method used

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  • Antibody, test strip and kit against bovine nodular skin disease virus
  • Antibody, test strip and kit against bovine nodular skin disease virus
  • Antibody, test strip and kit against bovine nodular skin disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Prokaryotic expression and purification of P32 protein

[0035] In this example, the prokaryotic expression and purification of P32 protein are carried out, and the specific steps are as follows:

[0036] 1. Query the LSDV genome sequence from GeneBank, select the conserved gene LSDV-P32 through alignment analysis, and express the P32 protein. The amino acid sequence of P32 protein was analyzed by the online software TMHMM, and it was found that there was a transmembrane domain at the C-terminus. Because the transmembrane domain protein was insoluble, the C-terminal transmembrane domain was removed during expression and codon optimization was performed. The amino acid sequence of LSDV P32 protein is shown in Table 3.

[0037] Table 3: Amino acid sequence of P32 protein

[0038]

[0039] 2. Select the correctly sequenced pGEX-6P-2-N280 plasmid, transform it into BL21(DE3) competent cells, and cultivate overnight in a 37°C constant temperature incubator. ...

Embodiment 2

[0041] Example 2: Preparation of P32 protein monoclonal antibody

[0042] Eight 8-12-week-old female Balb / c mice were immunized with the P32 protein antigen purified in Example 1. After immunizing 3 times, the mice were collected orbital blood to obtain mouse serum, and ELISA was used to detect P32 in mouse serum. Protein antibody titer, if the titer is less than 10,000, boost immunization 1-2 times until the antibody titer is greater than 10,000.

[0043] The mouse splenocytes with antibody titer>10000 were fused with myeloma SP2 / 0 cells, and the fusion cells were screened by HAT selection medium, and the fusion cells were screened and subcloned by ELISA. Ascites, the antibody was purified by Protein A / G antibody purification column. The ELISA titer of the purified antibody was >1:128000, and the purity was >90%.

[0044] The cell line numbers corresponding to the two monoclonal antibodies are 701 and 901, respectively.

Embodiment 3

[0045] Example 3: Amplification and Sequence Determination of CDR Region Sequence of P32 Protein Monoclonal Antibody

[0046] The hybridoma cell lines 701 and 901 were recovered and cultured. When the cells grew to the logarithmic growth phase, the cell count was about 8×10. 7 cells / ml, cells were collected. Extract the total RNA of hybridoma cells according to the instructions of TaKaRa MiniBEST Universal RNA Extraction Kit, according to the reaction system shown in Table 4, incubate at 65 °C for 5 min, then quickly cool on ice, and then set the reaction system shown in Table 5 at 42 °C for 60 min; 70 ℃15min; 25℃1min for 1st-Strand cDNA synthesis reaction.

[0047] Table 4: 1st-Strand cDNA synthesis reaction mix

[0048] reagent Usage amount Oligo dT Primer (50μM) 1μL dNTP Mixture (10mM each) 1μL template RNA <5μg

RNase Free dH 2 O

Up to 10μL

[0049] Table 5: Reverse Transcription Reaction Solution

[0050] reag...

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Abstract

The invention relates to an antibody specifically binding to bovine nodular skin disease virus (Lumpy skin disease, LSD) or an active fragment thereof, a colloidal gold immunochromatographic detection test paper or a kit including the antibody, and a preparation method and application thereof. The detection test paper or kit of the invention is convenient to produce, has stable effect, no non-specific reaction, low coating amount, and is favorable for mass production. The bovine nodular skin disease virus detection method provided by the invention is easy to operate and has good reproducibility, realizes a breakthrough in the field of on-site rapid detection of LSD, and enriches the existing LSD detection method.

Description

technical field [0001] The invention relates to the field of immunity, in particular to an antibody against bovine nodular dermatosis virus, and a detection test paper and a kit for detecting bovine nodular dermatosis virus. Background technique [0002] Lumpy skin disease (LSD) is an acute and subacute contact infectious disease of cattle caused by bovine nodular skin disease virus (LSDV). %, and the case fatality rate can reach as high as 10%. LSDV belongs to the genus Goatpoxvirus of the Vertebratepoxvirus subfamily of the Poxviridae family, and has up to 96% homology to the genomes of Sheeppoxviruses and Goatpoxviruses. The disease is a notified disease (OIE 2014 edition list) of the World Organization for Animal Health (English: World Organization for Animal Health; French: Office international desépizooties, OIE), and is a kind of infectious disease determined by the "People's Republic of China's List of Entry Animal Quarantine Diseases" The occurrence of this diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08G01N33/569G01N33/558G01N33/577G01N33/58
CPCC07K16/081G01N33/56983G01N33/558G01N33/577G01N33/587C07K2317/565C07K2317/56G01N2333/065G01N2469/10
Inventor 崔贝贝杨卫丽李霆段佳慧许宏瑞高宏应天翼李岩松余涛
Owner 北京弘进久安生物科技有限公司
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