Recombinant protein for detecting toxoplasma gondii and encoding gene of recombinant protein

A gene and coding technology is applied in the field of detection of recombinant protein of Toxoplasma gondii and its coding gene, which can solve the problems of poor specificity and low sensitivity of detection of Toxoplasma gondii, and achieves improved detection specificity and sensitivity, clear components and low cost. Effect

Pending Publication Date: 2020-05-08
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For this reason, the embodiment of the present invention provides a recombinant protein and its coding gene for detecting Toxoplasma gondii, so as to solve the problems of poor specificity and low sensitivity in detecting Toxoplasma gondii in the prior art

Method used

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  • Recombinant protein for detecting toxoplasma gondii and encoding gene of recombinant protein
  • Recombinant protein for detecting toxoplasma gondii and encoding gene of recombinant protein
  • Recombinant protein for detecting toxoplasma gondii and encoding gene of recombinant protein

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1, design and synthesis of gene sequence and primer

[0037] Referring to the MIC2 gene sequence of Toxoplasma gondii (No.XM_002367433.1), the MIC3 gene sequence of Toxoplasma gondii (No.EU572718.1) and the SAG1 gene sequence (No.GQ253075.1) in Genbank, according to the codons of the E. coli expression system Preferential codon optimization, and use Linker connection (amino acid sequence is GGGGSGGGGSGGGGS) for concatenation, codon optimization comparison see figure 1 . The optimized gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. At the same time, EcoRI and XhoI restriction sites were added to the 5' and 3' ends of the synthetic gene to obtain the MIC2-MIC3-SAG1 tandem gene. The nucleotide sequence is as follows: As shown in SEQ ID NO:1, the amino acid sequence of its encoded protein is shown in SEQ ID NO:2.

Embodiment 2

[0038] Example 2, construction of pET-32a-MIC2-MIC3-SAG1 recombinant expression plasmid

[0039] Use EcoRI and XhoI to double-enzyme digest the vector pET-32a(+) and the optimally synthesized Toxoplasma gondii MIC2-MIC3-SAG1 tandem gene fragment respectively, and gel the linearized pET-32a(+) plasmid and the target gene fragment Recycle. use T 4 Ligase ligated the tandem gene fragment of Toxoplasma gondii MIC2-MIC3-SAG1 and digested the linearized plasmid pET-32a(+) to obtain a recombinant expression plasmid, which was named pET-32a-MIC2-MIC3-SAG1 recombinant expression plasmid , The pET-32a-MIC2-MIC3-SAG1 recombinant plasmid was digested by EcoRI / XhoⅠ, and a DNA fragment of about 5900bp and 948bp appeared, which was linearized with the enzyme-digested plasmid pET-32a(+) and MIC2-MIC3-SAG1 target gene The size of the DNA fragments matches, such as figure 2 shown. Perform PCR identification on the selected plasmid, and amplify a DNA fragment of 813bp, which is consistent w...

Embodiment 3

[0040] Example 3, construction of pET-28a-MIC2-MIC3-SAG1 recombinant expression plasmid

[0041] The plasmid pET-28a(+) and the optimally synthesized Toxoplasma gondii MIC2-MIC3-SAG1 tandem gene fragment were double-digested with EcoRI and XhoI respectively, and the linearized plasmid pET-28a(+) and the target fragment were gel recovered . use T 4 Ligase connects the Toxoplasma gondii MIC2-MIC3-SAG1 tandem gene fragment with the digested linearized plasmid pET-28a(+) to obtain a recombinant expression plasmid, which is named pET-28a-MIC2-MIC3-SAG1 recombinant expression Plasmid, the pET-28a-MIC2-MIC3-SAG1 recombinant plasmid was digested by EcoRI / XhoI to obtain DNA fragments of about 5300bp and 948bp, and the digested linearized plasmids pET-28a(+) and MIC2-MIC3-SAG1 The size of the DNA fragment of the target gene is consistent, and the selected plasmid is identified by PCR, and a DNA fragment of 813bp is amplified, which is consistent with the size of the DNA fragment of th...

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Abstract

The embodiment of the invention discloses a protein which is a protein of the following a) or b): a) a protein consisting of amino acid sequences shown in SEQ ID NO:2, and b) a protein which consistsof amino acid sequences that amino acid sequences shown in SEQ ID NO:2 are substituted and / or deleted and / or added by one or more amino acid residues, and is specifically combined with antiserum of the toxoplasma gondii. According to the embodiment of the invention, genes of MIC2, MIC3 and SAG1 of the toxoplasma gondii are subjected to antigenicity analysis, a high antigenicity area is cut, codonoptimization is implemented according to preference of escherichia coli codon, a Linker (of which the amino acid sequence is GGGGSGGGGSGGGGS) is added for serial connection, a recombinant expression vector and a recombinant expression bacterium are constructed, MIC2-MIC3-SAG1 recombinant expression proteins of the toxoplasma gondii are obtained, and meanwhile, the defect that the yield of the expression protein is low is overcome.

Description

technical field [0001] The embodiment of the present invention relates to the field of biotechnology, in particular to a recombinant protein and its coding gene for detecting Toxoplasma gondii. Background technique [0002] Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii infection that can cause abortion, deformity or stillbirth in humans and animals. It is prevalent worldwide and seriously endangers public health and safety. Livestock production. Pregnant women, cancer patients, AIDS patients and other infections are particularly serious. Pregnant women infected with toxoplasmosis can lead to miscarriage, stillbirth, fetal malformation, and even fetal growth retardation and mental retardation. In addition, organ transplant patients and AIDS patients and other immunocompromised patients infected with Toxoplasma gondii can lead to death. [0003] Dogs and cats play an important role in the life history of Toxoplasma gondii. As the final host of Tox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/45C12N15/30C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/45C12N15/70G01N33/56905C12N2800/22G01N2333/45
Inventor 贾红王召阳刘雪婷鑫婷姜一曈朱鸿飞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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