A schistosoma japonicum antibody detection kit with red fluorescence activity detection protein

A red fluorescent protein and antibody detection technology, which is applied in the field of immunoassay, can solve the problems such as the sensitivity needs to be improved, and achieve the effect of high practical value, simple preparation and high sensitivity

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

However, immunochromatographic test strips can only be used for qualitative experiments, and the sensitivity of detection needs to be improved, while fluorescent immunochromatographic test strips can be used for qualitative or semi-quantitative detection, and have higher sensitivity

Method used

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  • A schistosoma japonicum antibody detection kit with red fluorescence activity detection protein
  • A schistosoma japonicum antibody detection kit with red fluorescence activity detection protein
  • A schistosoma japonicum antibody detection kit with red fluorescence activity detection protein

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Experimental program
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Effect test

Embodiment 1

[0035] The construction of embodiment 1 recombinant protein G

[0036] 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.

[0037][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.

[0038] [2] Kang Zeran. Cloning, prokaryotic expression characteristics and biological information analysis of monomeric red fluorescent protein gene DsRed2[D]. Yanbian University, 2016.

[0039] The fusion protein C3-RFP expressing recombinant G protein and red fluorescent protein was obtained by means of PCR, the nucleotide sequence of the fusion protein C3-RF...

Embodiment 2

[0041] Expression and purification of embodiment 2 recombinant protein G

[0042] 2.1 Expression of recombinant plasmids

[0043] phase

[0044] (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.

[0045] (2) When growing to the logarithmic phase (OD600 is about 0.6), add IPTG with a final concentration of 1mmol / 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)

[0046] Massive expression:

[0047] (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.

[0048] (2) When g...

Embodiment 3

[0060] Example 3 Activity identification of C3-RFP recombinant protein

[0061] 3.1 Observation of fluorescence activity

[0062] 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.

[0063] 3.2 Western blotting to detect the binding activity of recombinant protein and IgG

[0064] (1) Perform SDS-PAGE electrophoresis on the purified protein, then transfer the p...

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Abstract

The present invention provides a Schistosoma japonicum antibody detection kit with red fluorescence activity detection protein, which includes a Schistosoma japonicum antibody detection reagent strip and fusion protein C3-RFP; further, the present invention In the fluorescent test strip for Schistosoma japonicum antibody detection, soluble antigen (SEA) and streptococcal protein G (SPG) protein of Schistosoma japonicum were used as the coating antigen of the detection line (T) and quality control line (C) respectively. The test strip uses nitrocellulose membrane (CN95) as the chromatographic material, and the glass cellulose membrane as the sample pad, which is assembled into a test strip together with a PVC bottom plate and absorbent paper.

Description

technical field [0001] The invention relates to the field of immunodetection, in particular to a schistosoma japonicum antibody detection kit with a detection protein having red fluorescent activity. Background technique [0002] Schistosomiasis is a serious zoonotic parasitic disease caused by schistosomiasis, which is still one of the public health problems worldwide. Schistosoma infection can cause morbidity and death in humans and animals, affecting the health of humans and livestock and thus causing great economic losses. Schistosomiasis mainly causes organ damage and malnutrition and complications after infection. Studies have shown that the disability-adjusted life span caused by infection with schistosomiasis is almost equal to that of AIDS, and exceeds that of malaria and tuberculosis. The harm of schistosomiasis can be seen. Livestock are the main reservoirs of schistosomiasis. Studies have shown that the infection rates of domestic animals and humans are closely...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/58G01N33/533G01N33/543G01N33/558G01N33/68
CPCC07K14/315G01N33/6854G01N33/533G01N33/54313G01N33/588G01N33/558C07K2319/60C12N2800/22
Inventor 朱传刚柴瑞沈元曦纪荣毅林矫矫洪炀马以桐
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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