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Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same

A bovine rotavirus, protein antigen technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, viral peptides, etc., can solve the problems of inability to quickly diagnose, cumbersome process, time-consuming, etc., and achieve a time-saving diagnostic method. Effect

Inactive Publication Date: 2009-02-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that there is no effective prevention and treatment method for calf rotavirus diarrhea at present, and the diagnosis method using virus isolation has the disadvantages of time-consuming, laborious and incapable of rapid diagnosis, and the RT- The PCR method process is cumbersome and requires expensive molecular biology reagents and corresponding instruments and equipment, and provides a bovine rotavirus recombinant VP6 protein antigen and its preparation method

Method used

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  • Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same
  • Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same
  • Bovine rotavirus recombinant VP6 protein antigen and method of preparing the same

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specific Embodiment approach 1

[0015] Embodiment 1: In this embodiment, the recombinant VP6 protein antigen of bovine rotavirus is expressed by the Escherichia coli Rosetta (DE3) strain transformed with the recombinant vector plasmid pProEXHTa; wherein the VP6 gene of the bovine rotavirus NCDV strain is cloned in the recombinant vector plasmid pProEXHTa.

[0016] In this embodiment, the molecular mass of the bovine rotavirus recombinant VP6 protein antigen protein is about 50 ku, and can stimulate a strong immune response.

specific Embodiment approach 2

[0017] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the gene sequence of the complete open reading frame of the bovine rotavirus recombinant VP6 protein antigen is shown in SEQ ID NO:1. Others are the same as the first embodiment.

specific Embodiment approach 3

[0018] Specific embodiment three: the bovine rotavirus recombinant VP6 protein antigen of this embodiment is prepared according to the following steps: 1. Extract the viral nucleic acid of bovine rotavirus NCDV strain, and then carry out viral genome with the downstream primer P2 of the VP6 gene of bovine rotavirus NCDV strain Synthesis of cDNA, then carry out the PCR amplification of the VP6 gene with the upstream primer P1 and the downstream primer P2 of the VP6 gene of the bovine rotavirus NCDV strain; two, the VP6 gene subcloning obtained by the amplification is carried out in the pMD18-T vector, and then passed Cloning into the prokaryotic expression vector plasmid pProEXHTa after double digestion with BamHI and SalI to obtain the recombinant plasmid pPro-VP6; 3. Transform Escherichia coli Rosetta (DE3) with the recombinant plasmid pPro-VP6 to obtain the recombinant strain pPro-VP6 / Rosetta (DE3) 4. Induced expression of recombinant VP6 protein antigen; 5. Purification to o...

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Abstract

The present invention relates to a bovine rotavirus recombinant protein antigen and a preparation method thereof, in particular to a bovine rotavirus recombinant VP6 protein antigen and a preparation method thereof. The bovine rotavirus recombinant VP6 protein antigen resolves the following problems: at present, the method for diagnosing bovine rotavirus diarrhea needs a great deal of time and labor and cannot rapidly diagnose bovine rotavirus diarrhea; the process is complex; and expensive molecular biology reagents and corresponding instruments need to be used. The bovine rotavirus recombinant VP6 protein antigen is expressed by colibacillus converted by recombinant vector plasmids. The preparation method includes the following steps: (1) RCR amplification of VP6 gene; (2) acquisition of recombinant plasmids; (3) acquisition of recombinant bacteria; (4) inducible expression of recombinant VP6 protein antigen; (5) purification. The present invention provides a diagnosis antigen for the serodiagnosis of bovine rotavirus diarrhea, and the diagnosis antigen has extremely strong specificity and accuracy. The diagnosis method, which uses the bovine rotavirus recombinant VP6 protein antigen and adopts fluorescent antibody test and serological tests such as enzyme linked immunosorbent assay, has the advantages of time and labor saving and rapidness and does not need expensive reagents and instruments.

Description

technical field [0001] The invention relates to a bovine rotavirus recombinant protein antigen and a preparation method thereof. Background technique [0002] Bovine Rotavirus (BRA), also known as Calf diarrhea virus, belongs to the genus Rotavirus of the family Reoviridae. Bovine rotavirus mainly infects calves aged 1 to 7 days, and can cause digestive tract dysfunction in calves. Clinically, it is characterized by vomiting, diarrhea, dehydration and acid-base balance disorders. Infected cattle often die, and the case fatality rate can reach 50%. Because bovine rotavirus has the characteristics of wide prevalence, high incidence and great harm, it has developed into a worldwide disease. [0003] The traditional diagnostic methods of bovine rotavirus are often based on epidemiology and clinical manifestations as the basis for initial diagnosis. However, since bovine rotavirus and bovine viral diarrhea virus (BVDV) are often co-infected, and it is difficult to distinguish ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/14C12N15/46C12N15/70
Inventor 李一经葛俊伟唐丽杰马广鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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