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Dual-function protein with IgG binding activity and biotin binding activity and ELISA kit thereof

A bifunctional protein and binding activity technology, which is applied in the field of bifunctional protein G-SA and its ELISA kit, can solve the problems of affecting the accuracy of experimental results and increasing experimental costs

Active Publication Date: 2019-12-27
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, the conventional BSA method also inevitably uses enzyme-labeled secondary antibodies. According to the primary antibodies of different species, the secondary antibodies also need to be adjusted accordingly, which increases the cost of the experiment and affects the accuracy of the experimental results.

Method used

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  • Dual-function protein with IgG binding activity and biotin binding activity and ELISA kit thereof
  • Dual-function protein with IgG binding activity and biotin binding activity and ELISA kit thereof
  • Dual-function protein with IgG binding activity and biotin binding activity and ELISA kit thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 recombinant protein

[0045] 1.1 Biomaterials

[0046] Escherichia coli BL21 was purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmids containing SA, SPG, and plasmid pET-28a(+) were kept in the laboratory.

[0047] SA corresponding literature: [1] Zi Jing, Zhang Yuejuan, Wan Yi. Expression and purification of core streptavidin in Pichia pastoris [J]. Chinese Journal of Biological Products, 2018, 31(05): 485-488 .

[0048]SPG corresponding literature: [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]. China Journal of Animal Infectious Diseases, 2015,23(05):46-52.

[0049] 1.2 Construction and synthesis of recombinant protein

[0050] According to the C fragment of spG and the sequence of the streptavidin gene, primers were designed using Primer5 software, and restriction sites were added to ...

Embodiment 2

[0063] Expression and purification of embodiment 2 recombinant protein

[0064] 2.1 Expression of recombinant plasmids

[0065] Phase:

[0066] (1) Pick an appropriate amount of BL21 puncture bacteria containing the target fragment, inoculate in 5ml LB liquid medium containing Kan+, place in a shaking incubator at 37°C, and culture at 250rpm.

[0067] (2) When growing to the logarithmic phase (OD600 is about 0.6), add IPTG with a final concentration of 0.1 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.

[0068] SDS-PAGE analysis showed ( Figure 4 ), the recombinant plasmid pET-28a(+)-C3-SA was successfully expressed in Escherichia coli BL21(DE3), and the expression level did not change significantly with time at 1-8h after induction with 1mmol / L IPTG, and expressed after 2h induction reached the highest level and stabilized. When the inducti...

Embodiment 3

[0086] Example 3 Recombinant protein activity identification

[0087] 3.1 Western blotting to detect the binding activity of recombinant protein and IgG

[0088] (1) Perform SDS-PAGE electrophoresis on the purified protein, then transfer the protein to NC membrane, 130mA, 75min.

[0089] (2) Soak the NC membrane in 5% skimmed milk powder diluted with PBST, and seal at room temperature for 2 hours.

[0090] (3) Wash the blocked NC membrane three times with PBST, 5 min each time.

[0091] (4) The NC membrane was incubated with HRP-labeled goat anti-mouse IgG, rabbit anti-goat IgG, mouse anti-rabbit IgG, donkey anti-goat IgG, and biotin (diluted with PBST 1:2000) as antibodies, and incubated for 1 h at room temperature.

[0092] (5) Wash the incubated NC membrane three times with PBST, 10 min each time.

[0093] (6) The NC membrane was developed with DAB two-component chromogenic solution kit, and after color development, it was washed with running water to terminate the react...

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Abstract

The invention belongs to the field of genetic engineering, specifically, provides a dual-function protein with IgG binding activity and biotin binding activity and an ELISA kit thereof, and particularly relates to a codon-optimized dual-function protein G-SA with the IgG binding activity and the biotin binding activity, wherein the codon-optimized dual-function protein G-SA comprises streptococcalprotein G(SPG) fragments and an SA protein. The ELISA kit provided by the invention has good specificity, sensitivity and stability and has good application prospects.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a bifunctional protein G-SA with IgG binding activity and biotin binding activity and an ELISA kit thereof. Background technique [0002] At present, there are three kinds of flukes that infect humans: Schistosoma japonicum distributed in Asia, Schistosoma mansoni in central Africa and Latin America, and Schistosoma haematobium in northern Africa. In China, only Schistosoma japonicum is prevalent, distributed in the Yangtze River Basin and 12 provinces in the south of my country. Most of the cities and autonomous regions are very important agricultural production bases in China. Schistosomiasis japonicum is a widely distributed and serious zoonotic parasitic disease, which seriously endangers the health of the people and the development of the country. [0003] Diagnosis techniques for schistosomiasis mainly include etiological diagnosis and immunological diagnosis. The etiological diag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N15/62G01N33/535G01N33/68G01N33/58
CPCC07K14/315C12N15/70G01N33/535G01N33/6854G01N33/6845G01N33/581C07K2319/00C12N2800/22
Inventor 朱传刚纪荣毅袁娜娜张霁月沈元曦林矫矫洪炀马以桐
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI