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Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof

A foot-and-mouth disease virus and recombinant protein technology is applied in the field of foot-and-mouth disease virus 2C3ABC recombinant protein and its preparation, protein and its preparation, and can solve problems such as prolonging the time for response and diagnosis of foot-and-mouth disease prevention and control.

Inactive Publication Date: 2015-07-22
吕宏亮 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, serological methods have been used to detect non-structural protein antibodies of foot-and-mouth disease virus to evaluate the virus prevalence of foot-and-mouth disease in animals, including colloidal bead hemagglutination method, enzyme-linked immunosorbent adsorption transfer spot method, and multiple luminescence method. These methods need to be used in the laboratory. Different instruments and equipment limit the use of these methods in the field and prolong the time for response and diagnosis of FMD prevention and control

Method used

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  • Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof
  • Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof
  • Foot-and-mouth disease virus 2C3ABC recombinant protein as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Gene cloning and expression of recombinant protein 2C3ABC (mu2C3ABC) Bacterial construction, high-level expression and purification

[0043] 1. Experimental method

[0044] 1.1 Construction of natural 2C3ABC ORF

[0045] Using the sequence of FMDV O / China / 99 (Genbank: No.AF506822) as a template, design primers for 2C3ABC gene amplification: 2C3ABC(+): 5′-CACCGGGGCACTCAAAGCACGTGACATCAAT-3′, 2C3ABC(-): 5′-AGCTAAGCTTAGTGGTGTGGTTCGGGGTCAA-3 ', the two ends of the 2C3ABC gene contain restriction sites BamHI and Hind III, and the amplified product was double-digested with BamHI and Hind III and connected to the cloning vector pGEM-T to prepare the recombinant plasmid pGEM-2C3ABC.

[0046] 1.2 Construction of mutant 2C3ABC (mu2C3ABC) ORF

[0047] The main amino acid of the 2C3ABC ORF 3C protease active site was changed from Cys at position 142 to Ser (the corresponding codon was mutated from TGC to AGC), and Cys at position 163 was changed to Gly (the corresponding...

Embodiment 2

[0070] The detection of foot-and-mouth disease nonstructural protein antibody in the vaccine of embodiment 2

[0071] Method: The stock solution of foot-and-mouth disease virus was inactivated with BEI at 25°C for 24 hours, and after preliminary purification, purification, and emulsification, the content of the purified antigen was 20-23g / L, and the effective dose was 10-20μg / ml. The agent used Montanide ISA 206 (Seppic, Paris, France), emulsified for 15 minutes at 30°C according to the instructions (50:50), placed at 4°C for 24 hours and then stirred for 10 minutes. The integrity of 146S particles in the vaccine was measured with 10-30% sucrose density Gradient and 260nm spectrophotometer detection, containing O, A, Asia Ⅰ antigen 10-20μg per dose.

[0072] Observation of the virulence of the attack virus: the attack virus O, A, and Asia Ⅰ were obtained from the Veterinary Drug Testing Institute of the Ministry of Agriculture of China. 7 TCID 50 / ml, 4×10 6 TCID 50 / ml an...

Embodiment 3

[0080] Embodiment 3 immunochromatographic strip detects foot-and-mouth disease nonstructural protein antibody

[0081] 1. Materials and methods

[0082] 1.1 Test material

[0083] Serum: 62 positive sera of FMDV O / Taiwan / 1997 strain experimentally infected 14 days later were used as susceptibility test, 254 sera of naturally uninfected pigs and 167 vaccinated pigs were used for specificity test, 96 of 254 sera One part of the serum came from specific pathogen-free pigs (SPF) pigs, and the rest came from market-bought pigs. The pigs were immunized with the current O-type pig vaccine. Six vesicular virus antisera were self-made to evaluate the possible cross-reaction between FMDV and porcine vesicular virus. 320 sera were experimentally infected with FMDV O / Taiwan / 1999 strain, at 0, 2, 4, 6, 8, 10, 14, 21, 28, 34 days after infection, used for chromatography strip method and other commercial Method comparison. Serum in the acute phase was from infected FMDV O / Taiwan / 1997 str...

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Abstract

The invention discloses a foot-and-mouth disease virus (FMDV) 2C3ABC recombinant protein as well as a preparation method and application thereof, and belongs to the field of pharmaceutical biotechnology. According to the invention, the mutation of the following sites is performed on the basis of an original FMDV 3C protease gene: Cys142-Ser, Cys163-Gly. FMDV recombinant protein 2C3ABC is expressed as an inclusion body in the bacterial cytoplasm, and is subjected to separation, denaturation, renaturation and multi-step purification, to obtain a complete and enzymolysis-free FMDV nonstructural protein mu2C3ABC, wherein the protein has solubility and complete antigenicity, and has a molecular weight of 72 kDa. The protein, as a diagnostic antigen, is prepared into chromatographic strips, has sensitivities of 98.4% and 100% respectively in the detection of FMDV experimentally infected pigs and naturally infection-free pigs, and has specificities of 100% and 98% respectively in the detection of naturally infection-free pigs and vaccine-immunized pigs. Compared with commercial kits Ceditest and UBI, the FMDV 2C3ABC recombinant protein has a high degree of consistency, can be used to distinguish infected animals and immunized animals, wherein K = 0.823 (p is smaller than 0.05).

Description

technical field [0001] The invention relates to a protein and its preparation method and application, in particular to a foot-and-mouth disease virus 2C3ABC recombinant protein, its preparation method and application, and belongs to the field of medical biotechnology. Background technique [0002] Foot-and-mouth disease (FMD) is a disease that is highly contagious to artiodactyls and seriously affects the global economy and trade. Its pathogen is foot-and-mouth disease virus (FMDV), which belongs to the picornavirus family The foot-and-mouth disease virus genus has been found to have seven serotypes O, A, C, Asia Ⅰ, SAT1, SAT2, and SAT3. It is popular in Asia, Africa, and South America, including China, Southeast Asia, and Taiwan. In the past 50 years in mainland China, type O, Asia I, and A types of foot-and-mouth disease were mainly prevalent. [0003] FMDV is a single-stranded linear RNA with about 8500 nucleotides. The genome consists of 5'-untranslation region (Untra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/09C12N15/70C12N15/42C07K16/10G01N33/68
CPCC07K14/005C07K16/1009C12N2770/32122G01N33/56983G01N33/68G01N2333/09
Inventor 张澍吕宏亮
Owner 吕宏亮
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