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A detection protein with red fluorescent activity and its application

A red fluorescence and fluorescent protein technology, which is applied in the field of detection proteins with red fluorescence activity, can solve the problems of affecting stability and inactivation of biological macromolecules, and achieves the effect of simple preparation process and high yield.

Active Publication Date: 2022-05-17
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The method of labeling active substances such as antibodies or G proteins such as red fluorescent protein involves chemical coupling, purification after coupling, dialysis, concentration and other cumbersome steps; and due to the characteristics of biological macromolecules, the coupling binding sites are diverse. Not only may cause the inactivation of biological macromolecules, but also affect the stability

Method used

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  • A detection protein with red fluorescent activity and its application
  • A detection protein with red fluorescent activity and its application
  • A detection protein with red fluorescent activity and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The construction of embodiment 1 recombinant protein G

[0048] 1.1 Biomaterials

[0049] Escherichia coli BL21 was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the plasmid pET-28a(+) was stored in the laboratory, and the bacterial liquid containing the C3 fragment[1] and the bacterial liquid containing the RFP sequence[2] were used for the experiment Room preservation.

[0050] [1] Xu Rui, Zhao Dengyun, Hong Yang, Lu Ke, Li Hao, Lin Jiaojiao, Feng Jintao, Xu Yumei, Zhu Chuangang. Domain remodeling, expression and identification of streptococcal protein G [J]. Chinese Journal of Animal Infectious Diseases ,2015,23(05):46-52.

[0051] [2] Kang Zeran. Cloning, prokaryotic expression characteristics and biological information analysis of monomeric red fluorescent protein gene DsRed2[D]. Yanbian University, 2016.

[0052] 1.2 Construction and synthesis of recombinant protein G gene sequence

[0053] According to the gene sequence of the C3 fragment, the prime...

Embodiment 2

[0063] Expression and purification of embodiment 2 recombinant protein G

[0064] 2.1 Expression of recombinant plasmids

[0065] phase

[0066] (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.

[0067] (2) (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)

[0068] Massive expression:

[0069] (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.

[0070] (2) W...

Embodiment 3

[0082] Example 3 Activity identification of C3-RFP recombinant protein

[0083] 3.1 Observation of fluorescence activity

[0084] Centrifuge the bacterial solution collected in the above phase at 10,000rpm for 5min, discard the supernatant, and add 200ul ddH 2 O resuspend the bacteria, after resuspension, pipette 10ul onto a clean glass slide, cover with a cover glass, excite with RFP excitation light under a fluorescent electron microscope, and observe whether the bacteria have red fluorescence. Fluorescence electron microscopy observations showed that ( Figure 4 ), the fluorescence of recombinant plasmid pET-28a(+)-C3-RFP successfully induced and expressed in E. coli BL21(DE3) increased with time at 1-8h, and the fluorescence reached the highest after 8h of induction and tended to be stable.

[0085] 3.2 Western blotting to detect the binding activity of recombinant protein and IgG

[0086] (1) Perform SDS-PAGE electrophoresis on the purified protein, then transfer the pro...

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Abstract

The present invention provides a detection protein with red fluorescent activity and its application, characterized in that the detection protein includes a streptococcal protein G (SPG) fragment and a fluorescent protein, and the streptococcal protein G (SPG) fragment is C3 of SPG segment, the fluorescent protein is an optimized red fluorescent protein, and the detection protein has dual activities of fluorescent activity and binding to antibodies of different species. The detection protein disclosed by the present invention can broadly bind human and various animal total IgG and IgG of different subclasses. High protein binding molecules.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a detection protein with red 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 to false positives; immunology ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63G01N33/68
CPCC07K14/315C07K14/43595G01N33/68C07K2319/60
Inventor 朱传刚沈元曦纪荣毅柴瑞林矫矫洪炀马以桐
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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