A kind of duck Tembusu virus e protein truncated protein and application

A technology of duck Tembusu virus and protein, which is applied in the direction of antiviral immunoglobulin, virus, viral peptide, etc., can solve the problem of unstable expression, and achieve the effect of high sensitivity, large sample size and good stability

Active Publication Date: 2019-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to some reported literature, the applicant constructed several plasmids of different lengths, and expressed the corresponding truncated proteins in the prokaryotic. It was found that some of them had no specific reaction with the duck-positive serum of duck Tembusu virus, and some expression levels were unstable. There is no relevant report about the expression of mature duck Tembusu virus E protein for detection of serum

Method used

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  • A kind of duck Tembusu virus e protein truncated protein and application
  • A kind of duck Tembusu virus e protein truncated protein and application
  • A kind of duck Tembusu virus e protein truncated protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Obtaining of E truncated protein of DTMUV DF2 strain:

[0050] (1) DTMUVDF2 chicken embryo virus, take 200 μL, add 800 μL of Trizol reagent, mix well, let stand at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 seconds, ice bath for 10 minutes, 4 ° C, 12000 rpm, centrifuge for 15 minutes, take 600 μL Put the supernatant into a clean EP tube, add an equal volume of isopropanol solution, mix gently, let stand at room temperature for 10min, 4°C, 12000rpm, centrifuge for 10min, discard the supernatant, add 1mL absolute ethanol to the EP tube, mix gently , 4°C, 7500rpm, centrifuge for 5min, discard the supernatant, dry at room temperature, add 13μL DEPC water to dissolve, and store at -20°C.

[0051] (1) reverse transcription:

[0052] Add components according to the following system: AMV reverse transcriptase 0.5 μL, 5 times AMV reverse transcription buffer 4 μL, RRI 1 μL, random primer 1 μL, dNTPS 2 μL, RNA template 11.5 μL, a total of 2...

Embodiment 2

[0070] Obtaining of hybridoma cell line 4A10:

[0071] (1) Three healthy ducks were taken, blood was collected from the carotid artery, placed at 37°C for 30 minutes, and then separated at 4°C overnight, and the serum was collected. Extract duck serum IgG with caprylic acid-ammonium sulfate method, the specific operation is as follows: centrifuge the collected duck serum at 4°C and 4000rpm for 3 minutes, take the supernatant, add 4 times the volume of 60mM / L sodium acetate solution of pH 4.5, add as you go Stir, add 25 μL of n-octanoic acid per 1 mL of the above serum, let stand at room temperature for 30 min, centrifuge at 12,000 rpm for 10 min at 4°C, take the supernatant, filter with filter paper, and adjust the pH to 7.4 after filtration. Add a final volume of less than or equal to 45% saturated ammonium sulfate solution while stirring, stir at room temperature for 30 minutes, centrifuge at 4000rpm at 4°C for 10 minutes, remove the supernatant, resuspend the precipitate wi...

Embodiment 3

[0081] Obtaining enzyme-labeled secondary antibodies

[0082] (1) Preparation and purification of mouse anti-duck IgG mouse ascites:

[0083] The hybridoma cell line 4A10 was taken out from the liquid nitrogen tank, revived, and expanded for culture. Three days to one week in advance, BALB / C female mice aged 8-10 weeks were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant per mouse. The expanded hybridoma cells were counted with a cell counting plate, according to 5*10 5 Each mouse was injected intraperitoneally, and after 7-10 days, the ascites of the mice was collected every day until the mice died. The collected ascites was centrifuged at 12000 rpm for 5 mins, the precipitate was discarded, thimerosal was added to the supernatant with a final concentration of 0.01%, and stored at -80°C after aliquoting. Purify according to caprylic acid-ammonium sulfate method to obtain 4A10 monoclonal antibody, ie mouse anti-duck IgG.

[0084] (2) HRP-labeled mous...

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Abstract

The present invention relates to the technical field of animal virology and animal infectious disease detection. Specifically disclosed are a duck Tembusu virus E protein truncated protein and applications. A protein of the amino acid sequence as represented by SEQ ID NO.2 as a coating antigen and a duck Tembusu virus serum reagent kit prepared with a hybridoma cell line of which the accession number is CCTCC No: C2017169 are utilized in testing the serum and egg yolk antibodies of a duck population infected by a wild-type virus; great specificity is provided; the sensitivity is high, positivity can still be detected even when a duck Tembusu virus-positive serum is diluted to 1 : 12800; compared with a conventional agar diffusion test, the compliance rate is high, operation is easy, test time is reduced, and applicable in simultaneous testing of a large volume of samples.

Description

technical field [0001] The invention belongs to the technical field of animal virology and animal infectious disease detection. In particular, it relates to a duck Tembusu virus E protein truncated protein and its application. Background technique [0002] Duck Tembusu virus DTMUV (Duck Tembusu Virus) is a single-stranded positive-sense RNA virus with an envelope. Clinically, it causes an infectious disease characterized by the death of ducks and the decline in egg production of laying ducks. According to existing research reports, the virus can infect chickens, ducks, geese, pigeons and other animals. In 2011, the first waterfowl epidemic prevention and control seminar of the Chinese Association of Animal Husbandry and Veterinary Medicine named the disease caused by the virus as Avian Tembusu Virus Disease (ATMUVD), and the disease caused by ducks was called Duck Tambu. Su virus disease. [0003] Duck Tambusu virus disease first broke out in East my country in April 201...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18G01N33/68G01N33/569C07K16/10C12N5/20C12R1/91
CPCC07K14/005C07K16/1081C12N2770/24122G01N33/56983G01N33/6893G01N2333/183
Inventor 金梅林孙小云李淑云范俊青姚蓉杨应立
Owner HUAZHONG AGRI UNIV
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