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Indirect ELISA process for discriminating livestock infected with foot-and-mouth disease virus and livestock immunologically vaccinated with foot-and-mouth disease vaccine utilizing nonstructural prot

A foot-and-mouth disease virus, an indirect technology, applied in the directions of biochemical equipment and methods, microbial determination/inspection, material inspection products, etc., can solve problems such as being unsuitable for live animal quarantine, and achieve the effect of facilitating mass production and purification

Inactive Publication Date: 2005-04-06
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, this method is no longer suitable for live animal quarantine, and new means are needed to accurately distinguish between immune animals and infected animals

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of Example 1 3ABC Gene

[0039] 1. Extraction of foot-and-mouth disease virus RNA

[0040] RNA was extracted directly from FMDV-infected BHK21 cells using the Broad Technology Total RNA Extraction Kit. The operation was carried out according to the instructions of the kit. Specific steps are as follows:

[0041] ① Take 250 μL of cytotoxicity and add it to a 1.5 mL Eppendorf tube, then add 750 μL Trizol, vortex and mix, and ice-bath for 10 min.

[0042] ② Add 200 μL of chloroform, shake vigorously for 5 s, place at room temperature for 10 min; centrifuge at 12000 r / min at 4 °C for 15 min.

[0043] ③ Transfer the aqueous phase into a new Eppendorf tube with 50 μL of isopropanol, invert to mix, place at room temperature for 10 min; centrifuge at 12,000 r / min at 4°C for 15 min.

[0044] ④Remove the supernatant, a white precipitate can be seen at the bottom of the tube, wash with 100 μL of 75% ethanol, centrifuge at 10,000 r / min for 10 min, discard the ethanol, a...

Embodiment 2

[0123] The construction of embodiment 2 recombinant expression vector

[0124] Enzyme digestion and recovery of the target fragment and pTriEx-4 Neo vector:

[0125] ①The sequenced pGEM-3ABC plasmid and pTriEx-4 Neo plasmid were digested with Sal I and Bgl II and Xho I and Bgl II respectively.

[0126] The enzyme digestion system (5μL) is as follows, the target gene tube is 10×buffer D 5μL, BSA 1μL, Bgl II 2.5μL, Sal I 2.5μL, H 2 O 24 μL, pGEM-3ABC plasmid 15 μL. Carrier tube: 5 μL of 10× buffer D, 1 μL of BSA, 2.5 μL of Bgl II, 2.5 μL of Xho I, H 2 O 24 μL, pTriEx-4 Neo vector 15 μL.

[0127] ② After centrifuging and mixing the enzyme digestion mixture, place it in a 37°C water bath for digestion for 3 hours. ③ Take 3uL of the enzyme digestion mixture for electrophoresis observation. After the enzyme digestion is complete, inactivate the endonuclease by incubating in a water bath at 65°C for 15 minutes, and electrophoresis the recombinant plasmid and vector digestion prod...

Embodiment 3

[0135] Induced expression of embodiment 3 recombinant expression plasmid

[0136] Inoculate 30 μL of the sequence-analyzed recombinant bacteria into 3 mL of 2×YT culture solution (containing 50 μg / mL carbenicillin) containing 1% glucose, and shake overnight at 37° C. Inoculate 200 μL of the overnight culture into 20 mL of 2×YT culture solution (containing 50 μg / mL carbenicillin) containing 1% glucose, shake vigorously to OD 600 When the concentration is 0.6-1.0, add IPTG to a final concentration of 1 mmol / L, induce expression at 37°C, collect the bacterial liquid at 4 hours, perform polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis, and detect the expression product. At the same time, uninduced bacterial liquid was used as a negative control.

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Abstract

This invention relates to a foot-and-mouth disease virus non-structural protein 3ABC expressed with gene engineering language ,which is to identify the infected animal with foot-and-mouth disease and the animals having injected with inactivated vaccine of the disease.

Description

field of invention [0001] The invention relates to an indirect ELISA method in the field of animal husbandry and veterinary medicine by using non-structural protein 3ABC of foot-and-mouth disease virus to identify livestock infected with foot-and-mouth disease virus and livestock immunized with inactivated vaccine of foot-and-mouth disease virus. Background technique [0002] Foot-and-Mouth Disease Virus (FMDV) is the pathogen that causes foot-and-mouth disease in cloven-hoofed animals. The disease is a highly contagious disease that can cause a worldwide pandemic. It has a serious impact on animal production and international trade of its products, so countries all over the world attach great importance to the prevention and control of the disease. [0003] There are many serotypes of foot-and-mouth disease virus, complex antigen structure, and extensive antigenic variation. After the immunized animals are exposed to the new virus, they can also form a recessive infection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/561G01N33/569G01N33/577G01N33/68
Inventor 谢庆阁刘在新曹轶梅卢曾军
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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