Peste des petits ruminants virus H-F fusion protein, biological material related to same and application of peste des petits ruminants virus H-F fusion protein

A biological material and biological technology, applied in Peste des petits ruminant virus H-F fusion protein and its related biological materials and application fields, can solve the problem that the diagnostic antigen cannot form an immune space epitope, the expression is insufficient in spatial structure, and cannot form a high-level protein structural issues

Active Publication Date: 2017-08-29
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression products of Peste des petits ruminants H and F proteins expressed in most studies usually exist in the form of inclusion bodies of insoluble monovalent proteins, and the expression of proteins with soluble H-F fusion activity has not been reported
The expression of monovalent inclusion body protein cannot form high-level protein structure and natural spatial conformation epitope due to insufficient expression and misfolding of spatial structure, resulting in that it cannot form a good immune spatial epitope as a diagnostic antigen
At the same time, the expression product in the monovalent inclusion body has no biological activity, so it needs to be denatured and refolded
Protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and the renaturation rate is often difficult to improve.
This is the main constraint limiting its application

Method used

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  • Peste des petits ruminants virus H-F fusion protein, biological material related to same and application of peste des petits ruminants virus H-F fusion protein
  • Peste des petits ruminants virus H-F fusion protein, biological material related to same and application of peste des petits ruminants virus H-F fusion protein
  • Peste des petits ruminants virus H-F fusion protein, biological material related to same and application of peste des petits ruminants virus H-F fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, soluble expression rmHF1

[0069] 1. Synthetic genes

[0070] This application has designed three kinds of PPRV H and F protein fusion genes, which are respectively the rmHF1-Y gene shown in the 4th-3558th position of SEQ ID No.1 and the rmHF1-Y gene shown in the 1st-3684th position of SEQ ID No.3 rmHF2-Y gene and rmHF1-W gene shown in positions 4-3558 of SEQ ID No.5. The difference in the nucleotide sequence between the rmHF1-Y gene and the rmHF2-Y gene is that the 5' end is different, and the 24th-3564th nucleotides of SEQ ID No.1 and the 150th-3690th nucleotides of SEQ ID No.3 are the same .

[0071] Both the rmHF1-Y gene and the rmHF1-W gene encode the protein rmHF1 shown in SEQ ID No.2, and the rmHF2-Y gene encodes the protein rmHF2 shown in SEQ ID No.3. The difference in amino acid sequence between rmHF1 and rmHF2 lies only in the amino terminal, and the amino acid residues 8-1184 of SEQ ID No. 2 and 51-1227 of SEQ ID No. 3 are the same.

[0072]...

Embodiment 2

[0088] Example 2. Using rmHF1 protein as the coating antigen indirect ELISA method to detect antibodies against Peste des petits ruminants virus

[0089] The relevant solutions in this embodiment are as follows:

[0090] The preparation of PBS buffer solution with a concentration of 0.01M and a pH value of 7.4: 8.5g NaCl, 0.2g KCl, 2.9gNa 2 HPO 4 12H 2 O, 0.59g NaH 2 PO 4 2H 2 O, 1L deionized water.

[0091] Coating buffer: 0.05mol / L sodium carbonate-sodium bicarbonate buffer (pH9.6), solvent is water, solute and its concentration are as follows: Na 2 CO 3 1.59g / L and NaHCO 3 2.93g / L.

[0092]Washing solution: 0.5% Tween washing solution. The 0.5% Tween washing solution was prepared as follows: Tween 20 and sodium azide were added to the PBS buffer solution with a concentration of 0.01M and a pH value of 7.4 until the content of sodium azide was 5 g / L, Tween 20 The content was 5mL / L, and 0.5% Tween washing solution was obtained.

[0093] Blocking solution: 1% BSA...

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Abstract

The invention discloses a peste des petits ruminants virus H-F fusion protein, a biological material related to the same and application of the peste des petits ruminants virus H-F fusion protein. The peste des petits ruminants virus H-F fusion protein is a protein a) or a protein b). The protein a) comprises amino acid sequences shown as SEQ ID No.2; the protein b) is a soluble protein, and each amino acid sequence shown as SEQ ID No.2 is substituted and / or deleted and / or added by an amino acid residue or a plurality of amino acid residues to obtain the soluble protein. The peste des petits ruminants virus H-F fusion protein, the biological material and the application have the advantages that indirect ELISA (enzyme-linked immunosorbent assay) detection methods for peste des petits ruminants virus antibodies are created by the aid of the peste des petits ruminants virus H-F fusion protein which is used as an envelope antigen, are high in specificity and sensitivity and good in accuracy and can be quickly, easily and conveniently implemented, and accordingly the peste des petits ruminants virus H-F fusion protein, the biological material and the application are favorable for clinically monitoring peste des petits ruminants.

Description

technical field [0001] The invention relates to the H-F fusion protein of Peste des Petits Ruminants virus and its related biological materials and application in the field of biotechnology. Background technique [0002] Peste des Petits Ruminants (PPR) is an acute, febrile, contagious disease caused by Peste des Petits Ruminants Virus (PPRV), which is characterized by high morbidity and mortality. At present, there is no effective method to treat Peste des petits ruminants. The only methods to control the disease are vaccination, culling after the outbreak, and strengthening regular serum monitoring. The most common reports at home and abroad use purified PPRV N and H proteins (full length) or partially recombinant N and H protein fragments containing the main antigenic epitopes of N protein as coating antigens, and there are also concentrated PPRV whole viruses as packaging antigens. An indirect ELISA method was established to detect PPR serum antibodies by the antigen (Q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/10C12N15/62C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/005C07K16/1027C07K2319/00C12N15/70C12N2760/18422G01N33/56983G01N2333/12
Inventor 孙雨宋晓晖杨林王传彬董浩杨天意
Owner CHINA ANIMAL DISEASE CONTROL CENT
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