Fluorescent protein marker preparation method and application

A fluorescent protein and protein technology, which is applied in the fields of molecular biology and genetic engineering, can solve problems such as the inability to guarantee repeatability between batches, and achieve the effect of ensuring accuracy

Inactive Publication Date: 2014-07-16
ZHENGZHOU IMMUNO BIOTECH
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AI Technical Summary

Problems solved by technology

At present, when performing SDS-PAGE, pre-stained markers (coupling through covalent bonds) can be used as tracer proteins, but the repeatability between batches cannot be guaranteed

Method used

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Examples

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Embodiment Construction

[0011] The preparation method of the present invention can be illustrated in detail by the following specific examples.

[0012] 1. Obtaining the sequence of the green fluorescent protein gene

[0013] Referring to the GFP sequence in NCBI (sequence number: ABE28521), its reading frame was synthesized and the restriction site NdeI-BamHI-SalI (5'→ 3') was added to the 5', and the restriction site ECORI-HindIII was added to the 3' -XhoI (5' → 3').

[0014] 2. Expression and purification of 30KDGFP protein

[0015] Using conventional molecular biology methods, the GFP reading frame was inserted between NdeI and XhoI of the bacterial expression plasmid PET30A (novagen) to obtain the bacterial expression plasmid 30KDGFP-PET30A. The plasmid was routinely transformed into E. Coli BL21DE3 (novagen), and the expression of 30KDGFP recombinant protein was induced according to the method recommended by the manufacturer, and the rRVFN recombinant protein was routinely purified with H...

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Abstract

The invention discloses a fluorescent protein marker preparation method and application. Carriers containing different fusion tags are selected according to the size of needed protein markers, primer sequences are determined according to the design of plasmids and insertion sequences, and the length is controlled to be about 20 bp; enzyme cutting sites are determined according to the different plasmids and different GFP gene segment sequences; the constructed plasmids are normally converted respectively, the plasmids are induced according to a method recommended by a manufacturer to express GFP recombinant protein in different sizes and be purified, then the 28 KD-80 KD fluorescent protein markers can be obtained, SDS-PAGE is adopted to detect the molecular weight and the purity of protein, and normal protein is quantified for standby application. According to the fluorescent protein marker preparation method and application, the expression carriers containing GFP open frames and the fusion tags in different sizes are constructed through recombinant, the carriers are induced to conduct expression and be purified respectively, the green fluorescent protein markers are prepared after the GFP protein in different sizes are selected to be mixed according to the needs, the inter-batch repeatability can be completely guaranteed and the experiment accuracy can be guaranteed.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to the preparation of a fluorescent protein marker, and also to the application of the fluorescent protein marker. Background technique [0002] Proteome refers to all proteins expressed by a genome, or a cell or tissue. The concept of proteome differs from that of genome in many ways, and it changes with different tissues and even environmental conditions. During transcription, a gene may be spliced ​​in multiple forms of mRNA, and the same protein may be post-translationally modified in many forms. Therefore, a proteome is not a direct product of a genome, and the number of proteins in a proteome can sometimes exceed that of a genome. Proteomics (Proteomics) is in an early "development" state, and experts in this field deny that it is a pure methodology. Like genomics, it is not a closed, conceptualized and stable body of knowledge, but a field. Proteom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66G01N27/447
Inventor 赵巧辉李桂林郭立攀李艳姣付光宇吴学炜
Owner ZHENGZHOU IMMUNO BIOTECH
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