Recombinant avian flavivirus E protein and application thereof

A poultry yellow and virus technology, applied in the field of genetic engineering, achieves the effects of good safety, good antigenicity and simple operation

Active Publication Date: 2014-04-16
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have strong specificity and good sensitivity, but these methods require expensive equipment and reagents, and require skilled operators
Not only is it inconvenient to test a large number of clinical samples, but it is also not conducive to the use and promotion of grassroots units
Indirect ELISA method can be used for serological detection of antibodies. Using purified whole virus as the coated antigen has the disadvantages of low virus culture yield, difficult purification, and high production cost, which is not conducive to the research and development of commercial diagnostic kits.

Method used

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  • Recombinant avian flavivirus E protein and application thereof
  • Recombinant avian flavivirus E protein and application thereof
  • Recombinant avian flavivirus E protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Preparation of recombinant fowl flavivirus E protein

[0042]1.1 Design and synthesis of specific primers

[0043] Using the Primer Premier5.0 primer analysis software, a pair of specific primers were designed according to the E gene sequence in the complete gene sequence of the fowl flavivirus JS804 strain (GenBank accession number: JF895923) collected by GenBank. Bam HI restriction endonuclease site, set in the downstream primer Eco RI restriction endonuclease site, the estimated length of the amplified fragment is 1444bp. The primer sequences are: upstream primer: 5'-GTGGATCCATGCAGAACCGAGAC-3' (SEQ ID NO: 3), downstream P2: 5'-GTGAATTCAATGGATCTGTCCCT-3' (SEQ ID NO: 4). Both upstream and downstream primers were synthesized by Nanjing GenScript Company.

[0044] 1.2 Cloning of the E gene of fowl flavivirus

[0045] Using Axygen’s bodily fluid virus total RNA extraction kit, directly from infected poultry flavivirus JS804 strain (Huang Xinmei, Li Yi...

Embodiment 2

[0058] Embodiment 2 establishes indirect ELISA with recombinant fowl flavivirus E protein

[0059] The purified recombinant fowl flavivirus E protein was used as coating antigen to establish indirect ELISA, and the optimal protein coating concentration, serum dilution concentration and ELISA optimal reaction conditions of indirect ELISA were explored by using square array method. The final conditions are as follows:

[0060] 1. Coating: Dilute the purified recombinant E protein to the working concentration with coating buffer (0.05M carbonate buffer, pH 9.6), add to 96-well ELISA plate, 100 μL per well. The coating amount of recombinant fowl flavivirus E protein was 4.375 μg / well, coated overnight at 4°C, dried and washed twice with PBST (0.05M pH7.4 PBS + 0.05% Tween-20);

[0061] 2. Blocking: The blocking solution is PBST containing 1% bovine serum albumin (BSA), add 300 μL to each well, block at 37°C for 2 hours, spin dry, and wash 3 times with PBST;

[0062] 3. Serum r...

Embodiment 3

[0071] Embodiment 3 Indirect ELISA method performance detection experiment

[0072] 3.1 Specificity test

[0073] The indirect ELISA method established by recombinant poultry flavivirus E protein was used to detect positive sera of Newcastle disease, avian influenza, egg drop syndrome, infectious laryngotracheitis, infectious bursal disease, chicken infectious bronchitis, etc., each sample Do 2 repetitions and carry out cross-reactivity determination, and the results are all negative, indicating that the method has no cross-reaction with the above-mentioned virus antibodies.

[0074] 3.2 Repeatability test

[0075] Repeated test in the plate: the same ELISA plate coated with the optimal concentration of the antigen was tested 4 times for 5 positive samples and 5 negative samples, and standard positive and standard negative serum controls were set up;

[0076] Repeated test between plates: use 4 ELISA plates coated with the optimal concentration of antigen, test 5 positive ...

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Abstract

The invention discloses recombinant avian flavivirus E protein and application thereof, which relate to the field of genetic engineering. The recombinant avian flavivirus E protein is obtained by gene cloning and expression technology. An indirect ELISA (enzyme-linked immuno sorbent assay) method which is established by using recombinant E protein as coating antigen is used for detecting avian flavivirus antibody. The method is the ELISA method, which constructs the ELISA method establishing detection, and recombinant avian virus E protein is initially and internationally used to construct and detect the avian flavivirus antibody. The method has the advantages of low cost, high specificity, high flexibility and the like. The recombinant avian flavivirus E protein can be used for determining specific antibody level of the avian flavivirus in a clinical domestic fowl, and natural infection. The invention further provides application of the recombinant avian flavivirus E protein to preparation of monoclonal antibody, polyclonal antibody, vaccines and protein chips.

Description

Technical field: [0001] The invention relates to the field of genetic engineering. Specifically relates to a recombinant fowl flavivirus E protein and application thereof. Background technique: [0002] Avian Flavivirus (Avian Flavivirus) is a flavivirus newly discovered in China in 2010 that can cause decreased egg production, decreased feed intake, growth retardation and death of poultry. Many scholars in China have reported that the virus is pathogenic to ducks, geese, chickens, etc. 2): 354-360; Jingliang Su, Shuang Li, Xudong Hu, et al . Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related Flavivirus. PLoS One. 2011, 6(3); and death. Chinese Journal of Animal Bed Infectious Diseases, 2010, 18 (6): 1-4; Chen Shilong, Chen Shaoying, Wang Shao, etc. Isolation and preliminary identification of a new type of flavivirus that causes egg production decline in laying hens. Fujian Agricultural Journal, 2011, 26(2):170-174). As a new epidemic disease, the dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C07K14/18C12N15/70C12N1/21G01N33/68C07K16/10A61K39/12A61P31/14C12R1/19
Inventor 黄欣梅李银赵冬敏刘宇卓钮慧敏韩凯凯张敬峰
Owner JIANGSU ACAD OF AGRI SCI
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