Construction method, expression method and detection method for protein tag carrier

A construction method and protein technology, applied in the field of biomarkers, can solve problems such as difficulty, non-expression, and uncertainty of the length of the target gene promoter

Active Publication Date: 2019-03-01
ANHUI NORMAL UNIV
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Problems solved by technology

There are the following problems: First, the effective promoter length of the target gene is uncertain
If the promoter is too short, it may not be effective, and the GFP fusion protein will not be expressed, and if it is too long, it will be difficult to amplify

Method used

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  • Construction method, expression method and detection method for protein tag carrier
  • Construction method, expression method and detection method for protein tag carrier
  • Construction method, expression method and detection method for protein tag carrier

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Embodiment Construction

[0038] It should be noted that the following biotechnology terms are involved in the embodiments of the present invention:

[0039] (1) Marking of the last exon: firstly, the penultimate exon of a gene in the genome (here, the nanos gene, called the target gene) (here, the third exon of the nanos gene, Exon 3) downstream partial sequence", "the last intron (here is the 3rd intron of the nanos gene, Intron 3)", "the last exon (here is the 4th exon of the nanos gene , Exon 4) and its downstream 100bp sequence" were amplified and cloned, called the target sequence. Then, a tag sequence (such as GFP) is inserted in front of the stop codon (such as TAG) after the internal coding sequence of the exon at the end of the target sequence to obtain the modified target sequence. Finally, the modified target sequence is integrated into the genome through the carrier of the transgene, and the sequence can participate in the RNA splicing and protein translation of the target gene in the cel...

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Abstract

The invention discloses a construction method, expression method and detection method for a protein tag carrier and relates to the technical field of biomarking. With Drosophila ovarian germ cell specific highly-expressed nos gene taken as an example, a cellular endogenous protein marking technique is developed, namely a protein tagging technique based on terminal exon marking; first, a transgeniccarrier with GFP (green fluorescent protein) tag sequence embedded in the terminal exon is constructed for nos gene; second, Drosophila with the transgenic carrier is prepared by means of mature transgenic technology of Drosophila; whether the expression product of the nos gene in Drosophila ovary cells is subjected to GFP fluorescence expression is detected; the technique herein facilitates theexpression pattern research for model organism Drosophila; references are provided for mammalian gene expression and positioning.

Description

technical field [0001] The invention relates to the technical field of biomarkers, in particular to the construction of a protein tag carrier and its expression and detection methods. Background technique [0002] The process of RNA splicing is the process of excising non-coding regions called introns from mRNA precursor molecules and splicing the coding regions of genes called exons to form mature mRNA. It is a very important biological process in the gene expression of eukaryotic cells. Through RNA splicing, many functional mRNAs with coding information can be produced. It is very important for the development and evolution of organisms. The RNA splicing body is responsible for the RNA splicing process. Specifically, it cuts the RNA at the junction of the exon and the intron of the initial transcription product of the mRNA (Precursor mRNA, referred to as pre-mRNA), and then connects the exons in turn, cutting out Introns make up a complete mRNA. Studies have shown that t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/85A01K67/033G01N33/68
CPCA01K67/0339A01K2227/706A01K2267/03C12N15/65C12N15/85G01N33/68G01N2333/43595
Inventor 陈冬生陶小倩孙铭钟吴华勤
Owner ANHUI NORMAL UNIV
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