1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein

A duck hepatitis A virus and recombinant protein technology, applied in the field of bioengineering, to achieve good specificity and high coincidence rate

Active Publication Date: 2016-02-17
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bioinformatics analysis and speculation show that the capsid proteins of DHAV may be VP1, VP3 and VP0 instead of VP1, VP2, VP3 and VP4 like other picornaviruses, so whether DHAV is VP0 or VP2 and VP4, whether they are located in Exposure on the surface of the virus and its antigenicity have not been reported so far

Method used

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  • 1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein
  • 1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein
  • 1-type duck hepatitis A virus VP4 recombinant protein, ELISA kit and preparing method of 1-type duck hepatitis A virus VP4 recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A type 1 duck hepatitis A virus VP4 recombinant protein, the amino acid sequence of the type 1 duck hepatitis A virus VP4 recombinant protein is shown in SEQ ID NO:1.

[0046] A preparation method of type 1 duck hepatitis A virus VP4 recombinant protein, comprising the following steps:

[0047] Step 1: Obtain the target fragment of VP4: determine the cleavage site and specific primer of the VP4 truncated gene of duck hepatitis A virus type 1, and the upstream primer is 5'- GAATTC TACCAGTAGACTTTCATGCAATGG-3' (SEQ ID NO.2), the downstream primer is 5'- CTCGAG TTGAGCTCCTACTTCATAAGAACA-3' (SEQ ID NO.3), the stock solution of DHAV-1 virus strain stored in the laboratory was diluted 5 times with sterilized PBS, added 1 / 100 volume of double antibody, incubated at 37°C for 1h, centrifuged at 8000r / min for 5min, and taken The supernatant was inoculated with 9-11 day-old healthy duck embryos without maternal antibody to DHAV-1, discarded the dead embryos within 24 hours, collec...

Embodiment 2

[0091] Example 2 Analysis of expression form of VP4 recombinant protein and optimization of induced expression conditions

[0092] 1. Analysis of expression forms:

[0093] (1) Streak inoculate the correctly identified expressing bacteria on LB solid medium containing Amp, pick a single colony and rejuvenate overnight in LB liquid medium at 37°C, take 2mL of bacterial liquid to inoculate 100ml LB / Amp, and shake in a water bath at 37°C for 2.5~ 3h, to OD 600nm About 0.6.

[0094] (2) Add IPTG to a final concentration of 0.4mmol / L, and induce expression in a 37°C water bath for 4 hours.

[0095] (3) The bacterial solution was centrifuged at 4°C and 8000r / min for 10min, and the supernatant was discarded.

[0096] (4) Add 10mL of 20mM Tris-HCl with pH 8.0 to suspend the cells, under the condition of ice bath, sonicate for 30 sec / time, with an interval of 30 sec, several times until the bacterial liquid is clear, centrifuge at 4°C, 12000r / min for 10min, Collect the supernatant ...

Embodiment 3

[0109] An ELISA kit for detecting type 1 duck hepatitis A virus antibody, comprising an ELISA plate, a PBST buffer, a blocking solution, an enzyme-labeled secondary antibody, a chromogenic solution and a stop solution, and the ELISA kit also includes claim 1 The above-mentioned type 1 duck hepatitis A virus VP4 recombinant protein.

[0110] The blocking solution is PBS buffer containing 1% skimmed milk powder; the enzyme-labeled secondary antibody is HRP-labeled rabbit or goat anti-duck IgG dilution.

[0111] A method for preparing an ELISA kit, comprising the following steps:

[0112] Step 1: Coating: Type 1 duck hepatitis A virus VP4 recombinant protein was coated on an enzyme-labeled ELISA plate. The optimal coating concentration of the VP4 recombinant protein was 3.375 μg / ml, 100 μL / well, incubated at 37°C for 3 hours, and then transferred to Overnight at 4°C, the next day, wash the plate with PBST buffer 3 to 5 times, each time for 3 minutes, and pat dry;

[0113] Step ...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to 1-type duck hepatitis A virus VP4 recombinant protein, an ELISA kit and a preparing method of the 1-type duck hepatitis A virus VP4 recombinant protein. The amino acid sequence of the 1-type duck hepatitis A virus VP4 recombinant protein is shown as SEQ ID NO:1. The preparing method of the 1-type duck hepatitis A virus VP4 recombinant protein includes the following steps of obtaining VP4 target segments; constructing recombinant expression plasmids pET-32c-VP4; preparing the VP4 recombinant protein. The recombinant prokaryotic expression plasmids are successfully constructed, and soluble expression of the VP4 recombinant protein is successfully obtained and has good reactogenicity with rabbit anti-DHAV-1 serums, which shows that the VP4 protein of DHAV-1 successfully obtains prokaryotic expression; the ELISA kit for detecting the 1-type duck hepatitis A virus antibodies is built and provides test data and basic materials for detecting the DHAV-1 antibodies and further carrying out DHAV-1 related research.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a type 1 duck hepatitis A virus VP4 recombinant protein, an ELISA kit and a preparation method thereof. Background technique [0002] Duck Hepatitis A Virus (DHAV) can cause outbreaks of duck viral hepatitis in ducklings, which has the characteristics of acute onset, short course of disease, rapid transmission, and high mortality. Hemorrhage, also known as crooked neck disease. At present, almost all duck raising countries and regions in the world have brought relatively large economic losses to the duck raising industry. DHAV can be divided into three genotypes: DHAV-1, DHAV-2 and DHAV-3. DHAV-1 is type 1 duck hepatitis A virus, which is the most important pathogen causing duck viral hepatitis. It is the most widely distributed and has strong pathogenicity and high infection rate. The lethal rate to ducklings can be as high as 90%. At present, almost all du...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/10C12N15/70G01N33/569
CPCC07K14/005C12N2770/32422G01N33/56983G01N2469/20
Inventor 汪铭书程安春曹莹莹杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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