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Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit

A duck hepatitis virus, soluble technology, applied in the field of biochemistry, can solve problems such as unseen 3D proteins, and achieve the effects of good reproducibility, high coincidence rate and high sensitivity

Active Publication Date: 2015-01-21
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3D is a non-structural protein of DHAV. Although it is not a component of the virus particle, it plays an important role in the life cycle of the virus. It plays a leading role in the synthesis of negative-strand RNA and replicase, and its own enzyme Catalytic activity makes the 3D protein indispensable in the survival process of the virus. Therefore, 3D antibodies may be produced in the virus-infected body, but there is no report on whether the 3D protein can be used as an antigen to detect DHAV antibodies by ELISA; at the same time, high Purity and active antigens are applied to the preparation of ELISA detection kits, which are of great significance and promotion for the detection of duck viral hepatitis antibodies

Method used

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  • Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit
  • Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit
  • Application of soluble I-type duck hepatitis virus 3D protein to preparation of ELISA reagent and ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, cloning the 3D gene of type I duck hepatitis virus strain DHAV-H

[0026]Type I duck hepatitis virus strain H (DHAV-H) (GenBank: JQ301467), E. coli DH5α strain and pET-32(a)+ vector were all preserved and provided by the Poultry Disease Control Research Center of Sichuan Agricultural University. Various molecular biology reagents were purchased from Bioreagent Company.

[0027] According to the genome sequence of DHAV-H (GenBank: JQ301467), the primers for amplifying the 3D gene were designed, and the specific primers were as follows:

[0028] P1: 5'-gg ggtacc gatcaagggaaagtagtgagcaag-3' (SEQ ID NO.1), the underline indicates the Kpn Ⅰ restriction site;

[0029] P2: 5'-acgc ctcgag tcagatcatcatgcaagctgt-3' (SEQ ID NO.2), the underline indicates the Xho I restriction site; then the designed primers were synthesized by Treasure Bioengineering (Dalian) Co., Ltd.

[0030] Take the preserved DHAV-H virus solution and make 5-fold dilution with sterilized PB...

Embodiment 2

[0035] Embodiment 2, construction expresses the expression vector of type I duck hepatitis virus strain H 3D protein

[0036] The recovered DHAV-H-3D was ligated with the pJET1.2 vector, and the ligation reaction was carried out according to the instructions of the pJET1.2 cloning kit. The reaction system is shown in Table 2.

[0037] Table 2. Connection of DHAV-H-3D and pJET1.2 vector

[0038]

[0039] After mixing the system according to Table 2, perform transient centrifugation, and then connect at 20°C for 15 minutes. The ligation product was transformed into DH5α competent cells, and screened with Amp-resistant LB solid and liquid medium, and the screened colonies were detected by PCR using the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers, and the amplified products were Agarose gel electrophoresis, the results of figure 2 Shown in A. The results showed that positive clones were obtained by screening.

[0040] In order to further detect positive clone...

Embodiment 3

[0048] Embodiment 3, expression of type I duck hepatitis virus strain H soluble 3D protein

[0049] The expression host bacteria Escherichia coli (Escherichia coli) Rosetta, Escherichia coli BL21 and Escherichia coli BL21 (DE3) PLYS strains are all preserved by the Poultry Disease Control Research Center of Sichuan Agricultural University; the expression vector pET containing H 3D protein of type I duck hepatitis virus strain -32(a)+ / DHAV-H-3D was constructed from Example 2; various molecular biology reagents were purchased from Bioreagent Company.

[0050] The pET-32(a)+ / DHAV-H-3D plasmid obtained in Example 2 was transformed into BL21, Rosetta and BL21(DE3) PLYS expression bacteria respectively, and the transformed bacteria were placed in Amp-resistant LB liquid medium at 37°C and 120r Cultivate overnight under the condition of 1 / min, and expand the overnight culture and fresh Amp-resistant LB liquid medium at a volume ratio of 1:100 for about 3 hours the next day, and the O...

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Abstract

The invention discloses an application of a soluble I-type duck hepatitis virus 3D protein to preparation of an ELISA reagent and an ELISA kit. The ELISA kit comprises a soluble I-type duck hepatitis virus 3D protein coated reaction plate, an enzyme-labeled antibody, a developing solution and a stop solution. When being used for detecting I-type duck viral hepatitis antibodies, the ELISA kit is strong in specificity, good in reproducibility and high in sensitivity, and has a higher agreement rate than the ELISA kits taking totivirus as coating antigen in the aspect of detection, so that the ELISA kit can be used for the detection of clinical samples.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to the application of soluble type I duck hepatitis virus 3D protein in preparing ELISA reagents and an ELISA kit for detecting serum antibodies to type I duck virus hepatitis. Background technique [0002] my country is the country with the largest number of ducks in the world, and the duck breeding accounts for about 70% of the world's total. The duck industry occupies an important position in my country's agricultural economy. Duck Hepatitis Virus (DHV) can cause Duck Viral Hepatitis (DVH) in ducklings. DVH is a highly fatal infectious disease characterized by acute onset, short course of disease, high morbidity and mortality. [0003] Duck hepatitis virus belongs to the genus Avihepatovirus of the Picornaviridae family. The virus has three serotypes, namely DHAV-1, DHAV-2 and DHAV-3, and has no immune cross-reaction. In my country, type 1 duck viral hepatitis is mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/531G01N33/5767
Inventor 汪铭书程安春曹乾大陈孝跃
Owner SICHUAN AGRI UNIV
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