Recombinant multi-epitope antigen of bovine coronavirus and application thereof

A coronavirus and multi-epitope technology, applied in the fields of molecular biology and immunology, can solve the problems of genetic variation vaccine efficacy, economic loss and loss of cattle breeding, and achieve the goal of improving prokaryotic expression efficiency and preventing virus from infecting animals Effect

Active Publication Date: 2017-08-11
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once the disease occurs, it is very easy to cause mass infection and induce secondary infection, causing huge economic losses to the cattle industry characterized by intensive feeding
At present, the prevention and control of inactivated vaccines and live attenuated vaccines are mainly used clinically. However, there are problems such as biological hazards and genetic variation in inactivated vaccines and live attenuated vaccines, which lead to the loss of efficacy of the original vaccines.
However, there is no report on the bovine coronavirus epitope vaccine

Method used

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  • Recombinant multi-epitope antigen of bovine coronavirus and application thereof
  • Recombinant multi-epitope antigen of bovine coronavirus and application thereof
  • Recombinant multi-epitope antigen of bovine coronavirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Screening of BCoV S protein epitopes and design and synthesis of multi-epitope proteins

[0039]According to the literature reports on NCBI and the S gene sequence of bovine coronavirus epidemic strains published in the Genebank database, the recombinant antigenic epitope BCoV-double-S1-S2 was designed, and two well-conserved S proteins were selected at the same time. Antigen epitopes S1 and S2, S1 and S2 are repeated in series, and each epitope is connected by 4GS-4GS flexible amino acids. At the same time, the nucleotide sequence encoding the recombinant antigenic epitope BCoV-double-S1-S2 was optimized with reference to the Escherichia coli codon preference table to improve its prokaryotic expression efficiency. Finally, EcoR I and Not I restriction endonuclease sites and protective bases were added to both ends of the sequence of the recombinant antigenic epitope BCoV-double-S1-S2, and the recombinant antigenic epitope was synthesized by genetic engineeri...

Embodiment 2

[0050] Example 2: Construction and Identification of BCoV S Protein Antigen Epitope Recombinant Expression Vector

[0051] The prokaryotic expression vector pET-28a(+) and BCoV S protein antigen epitope concatenation were double-digested with EcoR I and Not I restriction enzymes, and the vector fragment and the target gene fragment were respectively recovered, and the target gene fragment was lysed with T4DNA ligase Connected to the pET-28a(+) vector, transformed into BL21(DE3) competent cells, picked a single colony identified as positive by double enzyme digestion, and sent it to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing.

[0052] The identification results of BCoV S protein antigen epitope recombinant expression vector construction are as follows: figure 2 shown by figure 2 It can be seen that M is a Marker with a molecular size of 10000bp, 1 is the map of the recombinant expression plasmid after enzyme digestion, the upper band is pET-28a(+), the size...

Embodiment 3

[0053] Embodiment 3: the establishment of BCoV indirect ELISA detection method

[0054] BCoV S protein recombinant antigen epitope induces expression of purified protein as antigen, coats 96-well plate with different protein concentrations of 0.1-10 μg / ml, uses BCoV positive bovine serum as primary antibody, and calf negative serum as primary antibody Anti-negative control, diluted according to the ratio of 1:20, 1:50, 1:80, 1:100, 1:200, 1:400, forming a matrix with protein dilution, HRP-rabbit anti-bovine IgG diluted 1:5000 As the secondary antibody, explore the optimal concentration of protein and primary antibody. Among them, the optimal concentration of protein coating is 0.30 μg / ml, and the detection effect is the best when the serum dilution concentration is 1:100. Preliminary establishment of BCoV indirect ELISA detection method.

[0055] Judgment result: the absorbance value of blank control and negative serum wells is less than 0.2, and the result is valid when the...

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Abstract

The invention discloses a recombinant multi-epitope antigen of bovine coronavirus, also discloses application of the recombinant multi-epitope antigen of the bovine coronavirus in bovine coronavirus prevention ad diagnosis and belongs to the field of molecular immunology. An amino acid sequence of the recombinant multi-epitope antigen of the bovine coronavirus is a concatemer of a bovine coronavirus BCoV S protein epitope. The recombinant multi-epitope antigen, capable of causing a neutralizing antibody, of the bovine coronavirus serves as an immunogen or a vaccine, the neutralizing antibody oriented to the bovine coronavirus BCoV can be produced after the animal body is immunized, and the BCoV can be neutralized in vitro to prevent the animal body from virus infection. In addition, the recombinant multi-epitope antigen of the bovine coronavirus can also serve as a reagent for detecting a bovine coronavirus BCoV antibody or a polypeptide antibody.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and immunology, in particular to a recombinant multi-epitope antigen of bovine coronavirus and its application. Background technique [0002] Bovine coronavirus (BCoV) is the causative agent of bovine diarrhea discovered in the 1970s and was first isolated in my country in 1985. Clinically, the main manifestations are neonatal calf diarrhea, bloody feces, and severe watery diarrhea (sometimes with blood and mucus) in adult cattle in winter. The disease is mainly transmitted through the digestive tract and respiratory tract, and the incidence rate is high. Once the disease occurs, it is very easy to cause mass infection and induce secondary infection, which will cause huge economic losses to the cattle industry characterized by intensive feeding. At present, the prevention and control of inactivated vaccines and live attenuated vaccines are mainly used clinically. However, there are pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/215A61P31/14G01N33/68G01N33/569C07K16/10C07K16/06
CPCA61K39/12A61K2039/552C07K14/005C07K16/06C07K16/10C07K2319/00C12N15/62C12N15/70C12N2770/20022C12N2770/20034C12N2800/22G01N33/56983G01N33/6854G01N2469/20
Inventor 程凯慧于志君王洪梅何洪彬杨宏军张亮沈付娆朱彤
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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