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Soluble Expression Method of Recombinant Peste des Petits Ruminants Virus h-f Fusion Protein

A PPR, protein technology, applied in microorganism-based methods, antisense single-stranded RNA viruses, viruses, etc., can solve the problem that the renaturation rate is difficult to improve, the diagnostic antigen cannot form immune space epitopes, and the expression amount is insufficient. Spatial structure And other issues

Active Publication Date: 2020-05-19
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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  • Soluble Expression Method of Recombinant Peste des Petits Ruminants Virus h-f Fusion Protein
  • Soluble Expression Method of Recombinant Peste des Petits Ruminants Virus h-f Fusion Protein
  • Soluble Expression Method of Recombinant Peste des Petits Ruminants Virus h-f Fusion Protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, soluble expression rmHF1

[0032] 1. Synthetic genes

[0033] 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 .

[0034] 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.

[0035] 2...

Embodiment 2

[0050] Example 2. Using rmHF1 protein as the coating antigen indirect ELISA method to detect antibodies against Peste des petits ruminants virus

[0051] The relevant solutions in this embodiment are as follows:

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Blocking solution: 1% BS...

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Abstract

The invention discloses a soluble expression method of a recombinantpeste des petits ruminant virus H-F fusion protein. The solubleexpressionmethod comprises a step of expressing an encoding gene of a protein in a living thing, thereby obtaining the protein, wherein the living thing is a microorganism, a plant or a non-human animal; the protein is a protein of a) or b), namely a) a protein consisting of amino acid sequences of SEQ ID No.2, and b) a soluble protein generated by substituting and / or deleting and / or adding one or more amino acid residues into an amino acid sequence of SEQ ID No.2. The recombinantpeste des petits ruminant virus H-F fusion protein treated with the solubleexpression method of the recombinantpeste des petits ruminant virus H-F fusion protein disclosed by the invention is high in expression level and low in production cost, and a basis is made for furtherdevelopment of commercial kits.

Description

technical field [0001] The invention relates to a soluble expression method of recombinant Peste des Petits Ruminants virus H-F fusion protein 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. The control and elimination of epidemic diseases is inseparable from effective vaccines and fast and sensitive detection methods as technical support. Therefore, increasing the research and development of fast, effective, and low-cost diagnostic testing reagents, and accelerating the promotion of related products to the market can provide my country's national small The implementation of the pest ruminant disease eradication plan provides strong technical support. [0003] The PPRV genome encodes six structural proteins, namely nucleoprotein (N), phosphoprotein (P), polymerase large protein (L), m...

Claims

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Application Information

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