Multispecies universal detection protein with green fluorescence activity and application of universal detection protein

A green fluorescence, protein technology, applied in the field of immunodetection, can solve the problems of biological macromolecular activity change, inactivation, green fluorescent protein fluorescence activity change, etc., and achieve the effect of simple preparation process and high-sensitivity detection

Inactive Publication Date: 2019-12-27
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The method of labeling active substances such as antibodies or G proteins such as green fluorescent protein involves chemical coupling, purification after coupling, dialysis, concentration and other cumbersome steps; and due to the characteristics of biological macromolecules, the coupling bindin

Method used

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  • Multispecies universal detection protein with green fluorescence activity and application of universal detection protein
  • Multispecies universal detection protein with green fluorescence activity and application of universal detection protein
  • Multispecies universal detection protein with green fluorescence activity and application of universal detection protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction of embodiment 1 recombinant protein

[0054] 1.1 Biomaterials

[0055] Escherichia coli BL21 was purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid pET-28a(+) was preserved in the laboratory, SPG (Xu Rui, Zhao Dengyun, Hong Yang, Lu Ke, Li Hao, Lin Jiaojiao, Feng Jin Tao, Xu Yumei, Zhu Chuangang. Domain Remodeling, Expression and Identification of Streptococcal Protein G[J]. Chinese Journal of Zoonotic Diseases, 2015,23(05):46-52.) EGFP corresponds to the serial number on NCBI: U55762

[0056] 1.2 Construction and synthesis of recombinant protein gene sequence (such as figure 1 B)

[0057] Detected from GenBank, the published gene fragments encoding EGFP and SPG region C, find out C1, C2, C3 region and D region, and obtain the gene fragments of C3 region. Analyze the signal peptide of the EGFP sequence to detect whether the gene sequence contains rare codons of Escherichia coli, that is, the frequency of use is less than 10%, and replac...

Embodiment 2

[0079] Expression and purification of embodiment 2 recombinant protein

[0080] 2.1 Expression of recombinant plasmids

[0081] phase

[0082] (1) Transfer the pET-28a(+)-C3-RFP recombinant plasmids with correct identification results into BL21(DE3), inoculate them in 5ml LB liquid medium containing Kan+, and place them in a shaking incubator at 37°C. Shake culture at 250rpm.

[0083] (2) When growing to the logarithmic phase (OD600 is about 0.6), add IPTG with a final concentration of 1 mmol / L to induce expression. Take 0.5ml bacterial liquid before induction and 1h, 2h, 4h, 6h, 8h after induction, and analyze the best induction time by SDS-PAGE electrophoresis. ( image 3 A,C,E)

[0084] Massive expression:

[0085] (1) Transform the pET-28a(+)-C3-RFP recombinant plasmids with correct identification results into BL21(DE3), inoculate them in 150ml LB liquid medium containing Kan+, and place them in a shaking incubator at 37°C. Shake culture at 250rpm.

[0086] (2) Whe...

Embodiment 3

[0098] Example 3 Activity identification of C3-RFP recombinant protein

[0099] 3.1 Fluorescence spectrum observation

[0100] The purified recombinant protein was prepared in different concentrations, and the excitation spectrum of the recombinant protein was scanned with a fluorescence spectrophotometer ( Figure 4 A), obtain the maximum excitation wavelength of the recombinant protein; use the maximum excitation wavelength to obtain the emission spectrum of the recombinant protein ( Figure 4 B). Observe the fluorescence intensity of the three recombinant proteins. It can be seen from the figure below that the fluorescence intensity of the three recombinant proteins is higher than that of the standard EGFP, among which the fluorescence intensity of C3-EGFP is the strongest, followed by C3DC3-EGFP, and C3DC3DC3-EGFP is the weakest, which may be related to the C3 region of the recombinant protein and the size of the EGFP fragment The C3 fragment in C3-EGFP is only one-thir...

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Abstract

The invention provides a multispecies universal detection protein with green fluorescence activity and application of the universal detection protein. The universal detection protein is characterizedin that the detection protein comprises a streptococcus protein G (SPG) segment and a fluorescent protein; and an IgG binding segment of an SPG gene is reconstructed, only a C3 region where the protein G can be specifically bound with the Fc terminal of an antibody IgG is retained, the C3 region is divided into three groups of C3, C3-D-C3 and C3-D-C3-D-C3, all the groups are connected to EGFP, anda prepared recombinant protein has the fluorescence activity and dual activity of binding to antibodies of different species. Disclosed evolutionary immunoglobulin binding molecules can widely bind to total IgG and IgG of different subclasses in humans and many animals in a wide spectrum mode, and compared with immunoglobulin binding molecules in the prior art, the binding force of the evolutionary immunoglobulin binding molecules is also higher than that of the existing immunoglobulin binding molecules.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a multi-species universal detection protein with green fluorescence activity and application thereof. Background technique [0002] The widespread spread of various infectious diseases poses a serious threat to my country's national security and population health. The development of sensitive and accurate pathogen analysis methods and detection technologies is of great significance for the rapid diagnosis and timely treatment of related diseases, the effective prevention and rapid treatment of bioterrorism and public health emergencies. The classic isolation and identification of microorganisms is cumbersome and time-consuming, and it is difficult to apply to the on-site rapid detection of pathogenic microorganisms; most of the detection methods based on pathogenic nucleic acids have high detection sensitivity, but require complex nucleic acid extraction processes, and are prone t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70G01N33/533C12R1/19
CPCC07K14/315C12N15/70G01N33/533C07K2319/60
Inventor 朱传刚纪荣毅沈元曦林矫矫洪炀岳永程
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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