Recombinant TsPKA-C protein and application thereof

A protein and amino acid technology, used in enzymes, biochemical equipment and methods, instruments, etc.

Inactive Publication Date: 2016-08-17
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above results undoubtedly indicate that cAMP-dependent protein kinase is expected to be used as a candidate antigen molecule for the diagnosis of porcine cysticercosis, thereby solving the problem of cross-reactivity in existing serological diagnostic methods

Method used

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  • Recombinant TsPKA-C protein and application thereof
  • Recombinant TsPKA-C protein and application thereof
  • Recombinant TsPKA-C protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] 1 Materials and Methods

[0024] 1.1 Strains and Serum

[0025] Positive and negative sera of Taenia solium adult worms and cysticerci suis were preserved by the Helminth Research Group of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0026] 1.2 Reagent

[0027] TRIzol Reagent, DNA gel recovery kit, pMD-T-19(simple) Vector, plasmid mini-extraction kit, restriction endonuclease Bam H I, Hin d III and T4 DNA ligase were purchased from Japan Takara Company. RevertAid First Strand cDNA Synthesis Kit and Prestained Protein Ladder were purchased from Thermo, USA. Escherichia coli DH(5α) competent cells, expression vector pET-30a, Escherichia coli BL21(DE3) competent cells, isopropyl-β-D-thiogalactoside (IPTG), X-Gal , 5× protein loading buffer were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Horseradish peroxidase (HRP)-labeled rabbit anti-pig IgG was purchased from Sigma. Ni Sepharose 6 Fast Flow protein...

Embodiment 2

[0067] Example 2 Based on recombinant antigen TsPKA-C Cysticercosis ELISA detection

[0068] Coat the enzyme-labeled reaction plate with 1 μg of purified TsPKA-C at 37°C for 1 h, the coating buffer is carbonate buffer solution with pH 9.6, the coating antigen concentration is 10 μg / ml, and the blocking solution is 1% BSA. After fully washed with PBST, add the doubling-diluted pig negative serum and porcine cysticercosis, porcine cysticercosis, porcine trichinella spiralis and porcine toxoplasma gondii positive serum, incubate at 37°C for 1 hour, wash with PBST three times, add 1:5000 times dilution The HRP-labeled rabbit anti-pig IgG was fully washed and then added with DAB for color development, and the absorbance was measured after the reaction was terminated with concentrated sulfuric acid. The test results are shown in Table 1.

[0069] Table 1 ELISA detection results using TsPKA-C as the coating antigen

[0070]

[0071] It can be seen from Table 1 that...

Embodiment 3

[0072] Example 3 Identification method of positive serum for Taenia solium

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Abstract

The invention provides a recombinant TsPKA-C protein and application thereof and particularly relates to application of the recombinant TsPKA-C protein as taenia solium/cysticercerci detection antigen and application of the recombinant TsPKA-C protein to preparation of a porcine cysticercosis detection kit. The recombinant TsPKA-C protein can specifically detect taenia solium/cysticercerci and has no cross reaction with porcine thin-neck cysticercerci, trichinella sui and the like.

Description

technical field [0001] The present invention relates to the recombinant TsPKA-C protein and its application, in particular to the application of the recombinant TsPKA-C protein as the detection antigen of Taenia solium / cysticercus and the application of the recombinant TsPKA-C protein in the preparation of the porcine cysticercosis detection kit. Background technique [0002] Taenia solium ( Taenia solium ) is an important zoonotic parasite, its larvae can not only parasitize the human brain, eyes, muscles, etc., causing cysticercosis in the corresponding parts, seriously endangering human health. Moreover, it can also parasitize the muscles, brains and other tissues of pigs, causing pork to be discarded and destroyed, resulting in large economic losses in the pig industry. It is a social, political and economic disease recognized worldwide. At present, most of the diagnostic methods for porcine cysticercosis are serological methods such as ELISA and EITB, but most of these...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12G01N33/573
CPCC12N9/12C12Y207/11011G01N33/573G01N2333/912G01N2800/26
Inventor 骆学农刘光学张少华郭爱疆郑亚东梁盼红侯俊玲才学鹏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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