1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof

A duck hepatitis A virus and recombinant protein technology, applied in the field of bioengineering, can solve the problem of little research on DHAV capsid protein

Active Publication Date: 2016-01-27
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The VP2 protein is located on the surface of the capsid, contains antigenic epitopes and some antigenic epitopes can induce the body to produce neutralizing antibodies, there is an interaction between the capsid protein VP2 and the pro-apoptotic protein / apoptotic precursor protein Siva, Coxsackie virus B3VP2 The protein may specifically bind to the apoptotic precursor protein Siva, thereby affecting the induction of apoptosis, the spread of the virus, and the pathological process caused by the virus, but there are relatively few studies on the DHAV capsid protein
Bioinformatics analysis and speculation show that the capsid proteins of DHAV may be VP1, VP3 and VP0, rather than VP1, VP2, VP3 and VP4 like other picornaviruses, so whether DHAV is VP0 or VP2 and VP4, whether they It is located on the surface of the virus and its antigenicity has not been reported so far

Method used

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  • 1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof
  • 1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof
  • 1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A type 1 duck hepatitis A virus VP2 recombinant protein, the amino acid sequence of the type 1 duck hepatitis A virus VP2 recombinant protein is shown in SEQ ID NO: 1.

[0047] A preparation method of type 1 duck hepatitis A virus VP2 recombinant protein, comprising the following steps:

[0048] Step 1: Obtain the target fragment of VP2: determine the cleavage site and specific primer of the VP2 truncated gene of duck hepatitis A virus type 1, and the upstream primer is 5'- GAATTC ACTCCTGTTCTTATGAAGTAGGAGC-3', downstream primer is 5'- CTCGAG CCTGATTGTCAAATGGTC-3', dilute the DHAV-1 virus stock solution stored in the laboratory 5 times with sterilized PBS, add 1 / 100 volume of double antibody, incubate at 37°C for 1 hour, centrifuge at 8000r / min for 5 minutes, take the supernatant and inoculate it for 9~ For 11-day-old healthy duck embryos without maternal antibody to DHAV-1, the dead embryos within 24 hours were discarded, and the allantoic fluid and embryo bodies of 2...

Embodiment 2

[0092] Example 2 Analysis of expression form of VP2 recombinant protein and optimization of induced expression conditions

[0093] 1. Analysis of expression forms:

[0094] (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.

[0095] (2) Add IPTG to a final concentration of 0.4mmol / L, and induce expression in a 37°C water bath for 4 hours.

[0096] (3) The bacterial solution was centrifuged at 4°C, 8000r / min for 10min, and the supernatant was discarded.

[0097] (4) Add 10mL of 20mM Tris-HCl suspension cells with pH 8.0, under the condition of ice bath, sonicate for 30 sec / time, with intervals of 30 sec for several times, until the bacterial liquid is clear, centrifuge at 4°C and 12000r / min for 10min, respectively Collect the s...

Embodiment 3

[0110] 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 VP2 recombinant protein.

[0111] The blocking solution is PBS buffer containing 5% skimmed milk powder; the enzyme-labeled secondary antibody is HRP-labeled rabbit or goat anti-duck IgG dilution.

[0112] A method for preparing an ELISA kit, comprising the following steps:

[0113] Step 1: Coating: Type 1 duck hepatitis A virus VP2 recombinant protein original coated enzyme-labeled ELISA plate, 100 μL / well, 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;

[0114] Step 2: Blocking: add 150 μL / well of PBST buffer containing 5% skimmed milk powder, block at 37°C for 1 hour, wash the plate according to the me...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to a 1-type duck hepatitis A virus VP2 recombinant protein, an ELISA kit and a preparation method thereof. The amino acid sequence of the 1-type duck hepatitis A virus VP2 recombinant protein is shown as SEQ ID NO:1. The preparation method comprises the following steps of: acquiring a VP2 target segment; constructing recombinant expression plasmid pProEx-HTb-VP2; preparing VP2 recombinant protein. The successfully-obtained VP2 recombinant protein is expressed in an insoluble inclusion body, the VP2 recombinant protein has good reactogenicity with rabbit-anti-DHAV-1 serum, and proved by the above, prokaryotic expression is successfully obtained by VP2 protein of DHAV-1; the ELISA kit for detecting 1-type duck hepatitis A virus antibody is established by the expressed VP2 recombinant protein, and provides test data and basic materials for detecting the DHAV-1 antibody and further performing relevant study of the DHAV-1.

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 VP2 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 the most important pathogen causing duck viral hepatitis. It is the most widely distributed and has strong pathogenicity and high infection rate. The lethality rate to ducklings can be as high as 90%. At present, almost all duck raising countries and regions in t...

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 程安春汪铭书曹莹莹杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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