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Modified duck circovirus Cap protein and preparation method and application thereof

A technology of duck circovirus and protein, applied in the direction of antiviral immunoglobulin, biochemical equipment and methods, viruses, etc., can solve the problems of limited application and inactivity, and achieve the promotion of immunogenicity, excellent effect and good application foreground effect

Active Publication Date: 2020-07-28
WEIFANG HUAYING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the protein prepared by this method does not form biologically active virus-like particles, and the expressed protein is an inactive inclusion body, which greatly limits its application.

Method used

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  • Modified duck circovirus Cap protein and preparation method and application thereof
  • Modified duck circovirus Cap protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Preparation of Transformed Cap Protein of Duck Circovirus and Obtainment of Engineering Bacteria

[0023] 1. DNAStar biological software analyzed the Cap protein sequence of duck circovirus SDFC12 strain (GenBank accession number: KY328304), and found that its N-terminal 21-36 peptide segment (RRFRRRRLRIARPRRR) is rich in arginine, which will seriously affect its ability in Escherichia coli Recombinant expression in expression systems. At the same time, the peptide of Yersinia pestis (TAKSKKFPSYTATYQF) is a T cell epitope with good cellular immune function, which can significantly improve the cellular immune function of various immunogens. Therefore, the present invention replaces the arginine-rich N-terminal 21-36 peptide of duck circovirus Cap protein with a T cell epitope peptide (TAKSKKFPSYTATYQF) to obtain a modified duck circovirus Cap protein, At the same time, in order to facilitate the nickel column purification of the subsequent modified protein, 6 histidines...

Embodiment 2

[0032] Preparation and Testing of Genetic Engineering Subunit Vaccine of Duck Circovirus

[0033] 1 bacterial strain

[0034] 1.1 The strain used for production is duck circovirus genetically engineered subunit vaccine production strain DP.

[0035] 1.2 Standards for strains used in production

[0036] 1.2.1 Morphological and biochemical properties

[0037]Cultivate overnight on the LB agar plate containing kanamycin, round, smooth colonies with neat edges, protrusions, milky white luster appear on the culture plate, and after Gram staining, they are Gram-negative short bacilli under the microscope; The biochemical test results were glucose fermentation+, indole test+, methyl red test+, VP-, citric acid utilization test-.

[0038] 1.2.2 Culture characteristics It can grow in the medium containing kanamycin.

[0039] 1.2.3 Identification test

[0040] 1.2.3.1 PCR detection Use the LB liquid culture of this bacteria as a template, and use the following PCR primers for PCR a...

Embodiment 3

[0077] Efficacy Test of Genetic Engineering Subunit Vaccine of Duck Circovirus

[0078] 1 Preparation of control vaccine

[0079] Referring to the preparation procedure of tissue-inactivated vaccines in the 2015 edition of "Chinese Veterinary Pharmacopoeia", duck circovirus WF strain was added in proportion to 10% formaldehyde solution, the final concentration of formaldehyde solution was 0.2%, and inactivated at 37°C for 12 hours. After the inactivation is complete, prepare duck circovirus tissue inactivated vaccines according to the method of 2.5 vaccine preparation part and 3 finished product inspection part in embodiment 2.

[0080] 2 Animal efficacy experiment

[0081] Sixty 7-day-old healthy susceptible ducks were equally divided into two groups, 20 in each group. The first group is the Cap protein vaccine immunization group, and the duck circovirus genetically engineered subunit vaccine prepared by the present invention is used for leg muscle immunization, 0.2ml / head....

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Abstract

The invention aims to provide a modified duck circovirus Cap protein as well as a preparation method and application thereof, that is, 21-36 peptide fragments rich in arginine at the N-terminal of theduck circovirus Cap protein are deleted, and a universal T cell epitope is introduced into a deletion region to promote immunogenicity; the amino acid sequence of the obtained modified duck circovirus Cap protein is SEQ ID NO: 1, and one nucleotide sequence is SEQ ID NO: 2. The modified duck circovirus Cap protein gene is connected with an expression vector and then transferred into escherichia coli BL21 (DE3), efficient soluble expression of duck circovirus Cap protein is achieved, the expressed protein is spontaneously assembled into virus-like particles, and the particles can be used for development of products such as duck circovirus gene engineering subunit vaccine and yolk antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a modified Cap protein of duck circovirus and its preparation method and application. Background technique [0002] Duck circovirus (Duck circovirus, DuCV) was first discovered in diseased duck tissues by German scholar Hattermann et al. in 2003. kind. At present, duck circovirus has been reported in Germany, Hungary, the United States, China Taiwan and other regions, and the existence of the virus was also detected in Fujian in my country in 2008. Duck circovirus can cause symptoms such as disheveled feathers, stunted growth, and weight loss in ducks. It can also infect ducks latently, invade the immune system of ducks, and cause the immune function of the body to decline. It is often associated with duck influenza virus, duck pox virus, and duck reovirus. Pathogens such as orphan virus, duck hepatitis virus, duck Escherichia coli and Riemerella anatipestifer formed a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C07K16/08C07K16/02A61K39/12A61P31/20
CPCC07K14/005C07K16/081A61K39/12A61P31/20C12N2750/10022C07K2317/11C12N2750/10034A61K2039/552A61K2039/5258Y02A50/30
Inventor 张勇王宏华王秀云曹阳王辉李佳礼焦绪娜辛瑞祥刘磊
Owner WEIFANG HUAYING BIOTECH CO LTD
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