Arabidopsis thaliana gamma-INF induced lysosome thiol reductase gene and application thereof

A reductase, technology of Arabidopsis thaliana, applied in the field of genetic engineering, to achieve strong sulfhydryl reductase activity and wide application

Pending Publication Date: 2017-10-27
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] According to the records of the three major international nucleic acid sequence databases of GenBank, EMBL and DDBJ, the GILT cDNA of humans and various animals has been cloned and studied to a certain extent, and the research on the Arabidopsis GILT gene, even the whole plant The research on GILT gene is still completely blank at home and abroad

Method used

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  • Arabidopsis thaliana gamma-INF induced lysosome thiol reductase gene and application thereof
  • Arabidopsis thaliana gamma-INF induced lysosome thiol reductase gene and application thereof
  • Arabidopsis thaliana gamma-INF induced lysosome thiol reductase gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Cloning of AtGILT cDNA

[0027] 1. Extract total RNA from Arabidopsis thaliana.

[0028] Use RNA extraction reagent (TIANGEN) to extract the total RNA of Arabidopsis thaliana according to its operating manual, and identify its purity by formaldehyde denaturing agarose gel electrophoresis, which is greater than 95%, and its concentration measured by ultraviolet spectrophotometer is 720ng / μL.

[0029] 2. Use Transgen cDNA reverse transcription kit to transcribe into first-strand cDNA.

[0030] 1) Primer design: Primers were designed according to the Arabidopsis thaliana database in NCBI and the human GILT sequence alignment: AtGILT-F1: 5'-ATGGCGTCGATGCCGAGCAAACTTC-3', AtGILT-R1 sequence: 5'-TCATAGCAGGCCGGTGATGTCGATG-3'.

[0031] 2) Reverse transcription: Add 3 μL of total RNA extract in sequence to a 1.5 mL Eppendorf tube treated with DEPC, Oligod(T) 18 (0.5μg / μL) 1μL, 2×TS Reaction Mix 10μL, DEPC water 5μL, TransScript RT / RI EnzymeMix 1μL; incubate at 42°C fo...

Embodiment 2

[0033] Gene copy number analysis and expression pattern analysis of embodiment 2AtGILT gene

[0034] 1. First use PCR to prepare probes, using the Arabidopsis genome as a template, the system is 50 μL, 10 μmol / L upstream and downstream primers (upstream primer: 5'-ATCTGATTATTCTGGGGTAT-3'; downstream primer: 5'-TGATGTCGATGTATGACG-3 ') each 1μL, 10×buffer(plus Mg 2+ ) 5 μL, dUTP labeling mixture 5 μL, Taq enzyme 15 μL, ddH2O 36 μL. The reaction program was 94°C for 5 min; 30 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 2 min; finally, an extension at 72°C for 7 min. After the reaction was completed, the product was separated by electrophoresis on a 1% agarose gel, and the DNA band was recovered with a gel extraction kit. Genomic DNA was extracted from Arabidopsis tissue samples and digested with Nco I and Xba I respectively. The system was buffer 60 μL, endonuclease 30 μL, genome 10 μg, ddH 2 O up to 600 μL, digested with an equal volume of phenol: chloroform and extr...

Embodiment 3

[0036] Example 3 Expression of Arabidopsis thaliana γ-INF-induced lysosomal thiol reductase polypeptide sequence by prokaryotic expression system

[0037] 1. First, use the previously constructed pMD19-T vector connected to the AtGILT gene as a template to perform PCR, the system is 25 μL, 10 μmol / L upstream and downstream primers (upstream primer: 5'-CGGGATCCGATTATTCTGGGGTATCTC-3'; downstream primer: 5'- CCGCTCGAGTCATAGCAGGCCGGTGATG-3') 1μL each, 2.5mmol / L dNTP 2μL, 10×DreamTaqbuffer 2.5μL, cDNA template 1.5μL, DreamTaq enzyme 0.3μL, add ddH 2 O 16.7 μL. The reaction program was: 94°C for 5min; 30 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 1min; finally, an extension at 72°C for 10min. After the reaction was completed, the product was separated by electrophoresis on a 1% agarose gel, and the DNA band was recovered with a gel extraction kit.

[0038] 2. Use BamH I and Xho I to perform double enzyme digestion experiments on the recovered DNA bands. The system is 1 μg ...

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Abstract

The invention discloses arabidopsis thaliana gamma-INF induced lysosome thiol reductase gene and application thereof. The arabidopsis thaliana gamma-INF induced lysosome thiol reductase has a DNA sequence as shown in SEQ ID NO.1, and the expression protein of the reductase has an amino acid sequence as shown in SEQ ID NO.2. According to the reductase, the cDNA complete sequence of the gamma-INF induced lysosome thiol reductase gene is cloned from plant arabidopsis thaliana, and lysosome thiol reductase protein is successfully expressed, the thiol reductase activity of the in-vitro expressed arabidopsis thaliana gamma-INF induced lysosome thiol reductase for human IgG is detected by adopting an in-vitro thiol protease activity detection method, and the result indicates that the protein has strong thiol reductase activity and can have wide application in the field of plant immune researches.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an Arabidopsis gamma-INF-induced lysosome sulfhydryl reductase (AtGILT) gene and application thereof. Background technique [0002] In 1988, Luster et al. discovered GILT and named it IP30. It binds to the mannose-6-phosphate receptor in the form of a soluble glycoprotein precursor, and after endocytosis, the N-terminal and C-terminal propeptides are cleaved to form a mature form of about 30kDa, which is then acidic in lysosomes It exerts sulfhydryl reducing activity in the environment and promotes the unfolding and further proteolysis of native protein antigens. GILT is mainly expressed in antigen-presenting cells, including monocytes / macrophages, B cells and bone marrow-derived dendritic cells, and can be induced by γ-INF in other cells such as fibroblasts and endothelial cells. In the endocytosis pathway, GILT is the only known enzymatically catalyzed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/10C12N15/63
CPCC12N9/0051C12N15/1096C12N15/63
Inventor 诸葛强桑明张嘉鑫陈燕
Owner NANJING FORESTRY UNIV
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