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VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein

A duck hepatitis A virus and recombinant protein technology, applied in the field of biomedicine, can solve the problems of low accuracy of duck viral hepatitis antibody, difficult expression and purification of VP0 protein, low detection efficiency, etc., and achieves good reactogenicity and strong specificity. , good repeatability

Inactive Publication Date: 2015-12-30
SICHUAN AGRI UNIV
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  • Claims
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Problems solved by technology

[0005] Therefore carry out prokaryotic expression for DHAV-1VPO gene, and the effect of the VPO protein of prokaryotic expression in detection and immune protection is studied, become the focus of the research of those skilled in the art, there is carrying out prokaryotic expression for DHAV-1VPO gene in the prior art Obtain the technology of VPO albumen, but this VPO albumen still exists and is difficult to express and purify, protein activity is low, the detection duck virus hepatitis antibody precision is not high, the shortcoming of low detection efficiency, thus it can be seen that the prokaryotic expression method of DHAV-1VPO gene With further improvement, the resulting VPO recombinant protein has large expression, easy purification, good protein activity, and has the advantages of high precision in the detection of duck viral hepatitis antibodies. It has become an effective way to detect and prevent duck viral hepatitis. Problems that need to be solved urgently by field personnel

Method used

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  • VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein
  • VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein
  • VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] A type 1 duck hepatitis A virus VPO recombinant protein, its amino acid sequence is shown in SEQ ID NO: 1, and its DNA sequence is shown in SEQ ID NO: 2.

[0054] A preparation method of type 1 duck hepatitis A virus VPO recombinant protein, comprising the steps of:

[0055] Step 1: Cloning of the whole gene of duck hepatitis A virus VP0 type 1: analysis and determination of the whole gene of duck hepatitis A virus type 1 VP0 and restriction sites, and design and amplification according to the whole gene sequence of DHAV-1X strain with Genbank accession number JQ316452.1 Specific primers for the DNA fragment of the VPO gene of duck hepatitis A virus type 1. At the same time, two enzyme cutting sites, BamHI and XhoI, were added to the 5' end of the primers to extract the RNA of duck hepatitis A virus type 1, and the specific primers were used for RT-PCR amplification. The target fragment of the whole gene of type 1 duck hepatitis A virus VPO was obtained.

[0056] Where...

Embodiment 2

[0101] The optimization of embodiment 2 recombinant protein expression conditions

[0102] experimental method:

[0103] 1. Optimization of IPTG concentration

[0104] Inoculate the expressing bacteria into four LB / Amp medium test tubes at a ratio of 1:100, and culture at 37°C until OD 600nm At 0.6, add IPTG to the final concentration: 0mmol / L, 0.4mmol / L, 0.8mmol / L and 1.2mmol / L to induce culture for about 4h, sample treatment, SDS-PAGE electrophoresis.

[0105] 2. Optimization of induction time

[0106] Inoculate the expressing bacteria into 5 LB / Amp medium test tubes according to the ratio of 1:100, and wait until OD 600nm After reaching 0.6, add the optimal IPTG concentration, respectively induce 4h, 6h, 8h, 10h and 12h, the treatment is the same as above.

[0107] 3. Optimization of induction temperature

[0108] Inoculate the expressing bacteria into 3 LB / Amp medium test tubes at a ratio of 1:100, and wait until OD 600nm After reaching 0.6, add the optimal IPTG conc...

Embodiment 3

[0111] Embodiment 3 Establishment of ELISA method based on recombinant protein

[0112] experiment method:

[0113] 1. Optimization of reaction conditions for recombinant protein-based ELISA method

[0114] The ELISA reaction conditions are operated according to the conventional method, and the optimization of the ELISA method reaction conditions is as follows:

[0115] (1) Antigen coating concentration: VP0 recombinant protein (1mg / ml) made 1:100, 1:200, 1:300, 1:400, 1:600, 1:800, 1:1200, 1:1600 and 1 : 2400 nine dilutions;

[0116] (2) Optimum dilution of serum: DHAV-1 positive / negative serum is 1:10; 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 seven dilutions ;

[0117] (3) The optimal dilution of the enzyme-labeled antibody: set five dilutions of 1:200, 1:400, 1:800, 1:1600 and 1:3200;

[0118] (4) Protein coating conditions: 37°C for 1h to 4°C overnight, 37°C for 4h to 4°C overnight, 4°C overnight and 37°C for 2h;

[0119] (5) Selection of blocking solution: set six b...

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Abstract

The invention discloses a VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as a preparation method and an application of the VP0 recombinant protein and belongs to the technical field of bio-medicine. The preparation method of the VP0 recombinant protein comprises the following steps: a), target fragment cloning of a whole VP0 gene and expression vector construction; b), expression of the VP0 recombinant protein; c), purification of the VP0 recombinant protein. The invention further provides research on the function of the VP0 recombinant protein in detection and immune protection. A novel way is provided for detection, prevention and treatment of duck viral hepatitis, an indirect ELISA (enzyme linked immunosorbent assay) method based on the VP0 recombinant protein is established, and the method has the characteristics of good repeatability and high specificity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a type 1 duck hepatitis A virus VPO recombinant protein and a preparation method and application thereof. Background technique [0002] Duck hepatitis, also known as duck viral hepatitis (Duckviral hepatitis, DVH), is a highly contagious and fatal duck viral infectious disease caused by duck hepatitis virus (Duckhepatitisvirus, DHV), which is characterized by acute onset, rapid transmission, The course of the disease is short and the mortality rate is high. The necropsy of sick ducks shows hepatomegaly, dendritic congestion or hemorrhagic spots on the surface. The disease was first discovered in the United States in 1945. DHV was initially divided into three serotypes (DHV-1, DHV-2, and DHV-3), and later DHV-2 and DHV-3 were classified into the Astroviridae family, and there was no antigenic correlation among the three serotypes. Now DHV-1 has been named duck hep...

Claims

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Application Information

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IPC IPC(8): C07K14/10C12N15/70G01N33/569A61K39/125A61P31/14
CPCA61K39/12C07K14/005C12N2770/32422C12N2770/32434C12N2770/32451G01N33/56983G01N2469/20
Inventor 程安春汪铭书齐晓燕杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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