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Trichina recombinant protein and application thereof

A technology of recombinant protein and trichinella, which is applied in the field of recombinant protein and application of trichinella, and can solve the problems of unsatisfactory diagnosis and the like

Active Publication Date: 2013-08-14
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

At present, the Trichinella spiralis antigens obtained by genetic engineering methods mainly include 46 kDa, 49 kDa, 53 kDa, etc. The recombinant antigens are reactogenic and can be recognized by the serum of infected animals, but the early diagnosis of Trichinella spiralis infection is still not ideal

Method used

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  • Trichina recombinant protein and application thereof
  • Trichina recombinant protein and application thereof
  • Trichina recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Extraction of total RNA from Trichinella spiralis

[0043] Extracted with Trizol (invitrogen), the operation is as follows:

[0044] (1) The mice infected with Trichinella spiralis for 35 days were dissected, the skeletal muscles were taken and crushed, and the muscle larvae of Trichinella spiralis were recovered by artificial digestion. About 100 mg of fresh worms were taken, placed in a micro-homogenizer, and added 1mL of Trizol, sonicated quickly in an ice bath until no visible tissue pieces.

[0045] (2) Transfer the liquid to a DEPC-treated microcentrifuge tube and let stand at room temperature for 5 min; add 0.2 mL of phenol-chloroform extract, shake vigorously for about 15 s, and incubate at room temperature for 2–3 min.

[0046] (3) After centrifugation at 12,000×g at 4°C for 15 min, transfer the supernatant liquid to another clean centrifuge tube, add 200 μL of chloroform and extract again.

[0047] (4) Transfer the extracted upper layer liquid to a clean ...

Embodiment 2

[0065] 1. Construction of prokaryotic expression pET30a-TsSerpin recombinant plasmid

[0066] pMD18-T-TsSerpin recombinant plasmid Eco R I and xho After I double enzyme digestion, cut the gel to recover the target gene fragment. The recovered target gene fragment and pET30a vector plasmid were respectively used Eco R I and xho I double enzyme digestion, the two enzyme digestion systems are: (1) 20 μL of the target gene fragment, 4 μL of 10× H buffer, Eco R I 2 μL, xho I 2 μL, BSA 2 μL, Triton 2 μL, sterilized double distilled water 8 μL; (2) vector plasmid 12 μL, 10× H buffer 4 μL, Eco R I 2 μL, xho I 2 μL, BSA 2 μL, Triton 2 μL, sterile double distilled water 16 μL. After the digestion mixture was centrifuged and mixed, it was digested in a water bath at 37°C for 4 h.

[0067] The digested product was recovered and purified with the gel recovery kit Agarose Gel DNA Extraction Kit, and then the product was ligated. The ligation system is: 7 μL of enzyme-digest...

Embodiment 3

[0072] 1. Purification of Recombinant Proteins

[0073] According to the method in Example 2, 1 L of bacterial liquid was co-induced, and the recombinant protein in the form of inclusion bodies was obtained by qualitative analysis. The inclusion body protein was purified by electroelution after gel cutting, and the protein content was measured by ultraviolet spectrophotometer. The formula is as follows:

[0074] Protein content (mg / mL)=(1.45×A 280 -0.74×A 260 )×dilution factor

[0075] The amount of recovered protein was determined to be about 6.58 mg of purified protein per 1 L of culture supernatant. After the purified TsSerpin recombinant protein was subjected to SDS-PAGE, there was only one obvious protein band at 48.5 kDa, and there were no other bacterial impurities, see Figure 4 .

[0076] 2. Western-blot identification of recombinant protein

[0077] The purified recombinant protein TsSerpin was first subjected to SDS-PAGE electrophoresis, and then transferred t...

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Abstract

The invention discloses a preparation method of a recombinant protein and in particular relates to a TsSerpin recombinant protein and application thereof. The preparation method of the TsSerpin recombinant protein comprises the following steps: carrying out reverse transcription, with total RNA (Ribonucleic Acid) extracted from pig trichina muscle larva as a template and Oligo(dT)18Primer as a primer; with the obtained reverse transcription Cdna (Deoxyribose Nucleic Acid) as a template and SEQ ID No.1 and SEQ ID No.2 as primers, carrying out PCR (Polymerase Chain Reaction) amplification, to obtain pig trichina TsSerpin gene; and carrying out the treatment including recombinant plasmid construction, escherichia coli conversion and the like to obtain target protein. The recombinant protein disclosed by the invention can be used for preparing an antigen for detecting trichinosis.

Description

technical field [0001] The invention relates to the field of molecular biology, and mainly uses the recombinant protein of Trichinella spiralis serine protease inhibitor gene TsSerpin for immunodiagnosis and vaccine development. The specific design includes the cDNA clone of the Trichinella spiralis gene TsSerpin and its prokaryotic expression in Escherichia coli BL21 (DE3) Expression and identification of reactogenicity, and segmental cloning, prokaryotic expression and identification of reactogenicity of TsSerpin were performed, and fragments with better reactogenicity were screened for immunodiagnosis. The recombinant protein was used in the mouse immune protection experiment to determine its immune protection. Background technique [0002] Trichinellois (Trichinellois) is caused by Trichinella ( Trichinella spp.) is a serious food-borne zoonotic parasitic disease that can spread widely among more than 150 species of animals, including humans. Infection from the meat o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/15C07K14/81C12N15/70
Inventor 付宝权盖文燕李文卉曲自刚谢志宙刘静宜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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