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Preparation method and use of soluble type-I duck hepatitis virus 3C protein

A duck hepatitis virus, soluble technology, applied in the field of biochemistry

Active Publication Date: 2018-01-19
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no effective drug for the treatment of DHAV clinically. After the onset, only hyperimmune serum and egg yolk antibody can be used for treatment.

Method used

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  • Preparation method and use of soluble type-I duck hepatitis virus 3C protein
  • Preparation method and use of soluble type-I duck hepatitis virus 3C protein
  • Preparation method and use of soluble type-I duck hepatitis virus 3C protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, cloning the 3C gene of type I duck hepatitis virus strain DHAV-H

[0030] 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.

[0031] According to the genome sequence of DHAV-H (GenBank: JQ301467), the primers for amplifying the 3C gene were designed, and the specific primers were as follows:

[0032] P1: 5'- catatg atgcaccatcatcatcatcatagcgggcgggtgaatttcagacata-3' (SEQ ID NO.1), the underline indicates the NdeI restriction site;

[0033] P2: 5'- aagctt taattgattaaaaactggaaagacccta-3' (SEQ ID NO.2), the underline indicates the Hind III restriction site; then the designed primers were synthesized by Treasure Bioengineering (Dalian) Co., Ltd.

[0034]Take the preserved DHAV-H virus solution and make 5-fold di...

Embodiment 2

[0039] Embodiment 2, construction expresses the expression vector of type I duck hepatitis virus H strain 3C protein

[0040] The recovered DHAV-H-3C was ligated with the pMD19-T simple vector, and the ligation reaction was carried out according to the instructions of the pMD19-T simple cloning kit. The reaction system is shown in Table 2.

[0041] Table 2. Connection of DHAV-H-3C and pMD19-T simple vector

[0042]

[0043] After mixing the system according to Table 2, perform transient centrifugation, and then connect at 16°C for 30 minutes. The ligation product was transformed into DH5α competent cells, and screened with Amp-resistant LB solid and liquid media, 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 subjected to agarose Gel electrophoresis, the results of figure 2 shown. The results showed that positive clones were obtained by screening.

[0044] In order to f...

Embodiment 3

[0052] Embodiment 3, expression of type I duck hepatitis virus H strain soluble 3C protein

[0053] The expression host bacterium E. coli BL21 bacterial classification is all preserved by Sichuan Agricultural University poultry disease prevention and control research center; Constructed in; various molecular biology reagents were purchased from Biological Reagent Company.

[0054] The pET-32(a)+ / DHAV-H-3C plasmid obtained in Example 2 was transformed into BL21 expression bacteria, and the transformed bacteria were cultured overnight in Amp-resistant LB liquid medium at 37°C and 120r / min, and the next day The overnight culture was expanded with fresh Amp-resistant LB liquid medium at a volume ratio of 1:100 for about 3 hours, and the OD of the bacterial solution was 600 When it reaches 0.6, add IPTG to the final concentration of 0.4mmol / L, and induce at 16°C for 12h, then collect the bacterial liquid, centrifuge the bacterial liquid for 10min under the condition of 12000r / min,...

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Abstract

The invention relates to a preparation method and use of a soluble type-I duck hepatitis virus 3C protein. The preparation method comprises connecting nucleotides at 3th to 574th sites shown in the formula of SEQ ID NO. 3 to polyclonal cleavage sites of a pET-32(a)+vector, carrying out activation in an Amp-resistant LB culture medium based on escherichia coli BL21 as host bacteria, and carrying out induced expression under conditions of the IPTG final concentration of 0.2-1.4 mmol / L and a temperature of 16-37 DEG C for 2-12h to obtain a soluble type-I duck hepatitis virus 3C protein. The protein is obtained by soluble expression, is natural, is free of denaturant-based denaturation and renaturation and can be used for 3C protease activity detection and 3C protease inhibitor screening. Thepreparation method has a great importance for research, development and screening of a drug for resisting type-I duck hepatitis viruses.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a preparation method of soluble type I duck hepatitis virus 3C protein, and also relates to an application of the soluble type I duck hepatitis virus 3C protein. Background technique [0002] my country is the country with the largest number of ducks in the world, and the number of ducks accounts for about 70% of the world. The duck industry occupies an important position in my country's agricultural economy, so the study of duck-derived microorganisms has important social and economic significance. Duck viral hepatitis is an acute and highly lethal infectious disease caused by duck hepatitis A virus (DHAV). Ducklings under the age of one week are a viral infectious disease that seriously threatens the duck industry. [0003] Duck hepatitis A virus belongs to the family Picornaviridae and the genus Avian Hepatitis Virus. 3C protein is one of the important proteases of Pi...

Claims

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Application Information

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IPC IPC(8): C07K14/10C12N15/70G01N21/64
Inventor 程安春孙迪汪铭书
Owner SICHUAN AGRI UNIV
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