Application of SPINK6 as marker for detecting AIDS

A marker, AIDS technology, applied in the field of biomedicine

Active Publication Date: 2020-09-22
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current related research is mainly about the occurrence and development of some diseases that SPINK6 inhibits the activity of the KLK (kallikrein) family, and the research on the function of SPINK6 and the inflammatory target LTA4H involved in disease regulation has not been reported.

Method used

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  • Application of SPINK6 as marker for detecting AIDS
  • Application of SPINK6 as marker for detecting AIDS
  • Application of SPINK6 as marker for detecting AIDS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Induced expression and activity identification of SPINK6 polypeptide

[0027] The sequence corresponding to SPINK6 was optimized according to the codons of Escherichia coli and the gene was synthesized to construct the PET-32a prokaryotic expression vector, and the expression host bacterium was BL21. Set enterokinase cleavage site. After the obtained glycerol bacteria were activated by streaking on the plate, the bacterial solution was shaken overnight at 37°C and 160rpm, and the expression was induced by IPTG at 28°C for 3-5h; SPINK6 was expanded overnight, and the bacteria were harvested, resuspended in Bindingbuffer and crushed. Using the method of affinity purification, using nickel column to purify the fusion protein, enterokinase digests the fusion protein, and selects the optimal digestion conditions (set the concentration of enterokinase as 0.5U / ml, in a 50 μL reaction system, set the added enterokinase The gradient of kinase is 1U, the concentration of SPINK6 ...

Embodiment 2

[0029]Detect the effect of SPINK6 on the binding and activity of LTA4H

[0030] (1) Combination experiment:

[0031] Surface plasmon resonance detection of the interaction of SPINK6 with LTA4H. LTA4H was coupled to the chip, and the mobile phase SPINK6 concentration was double-diluted (800nM, 400nM, 200nM, 100nM, 20μl / min, combined for 2min), and the binding curve was obtained by plotting with GraphPadPrism7.0 ( figure 1 In A), the result: there is a certain interaction between SPINK6 and LTA4H.

[0032] (2) Activity experiment:

[0033] The influence of SPINK6 on LTA4H epoxide hydrolase activity was detected by HPLC and Elisa method ( figure 1 B-D in ), the effect of SPINK6 on the aminopeptidase activity of LTA4H to hydrolyze its natural tripeptide substrate was detected by the ninhydrin method ( figure 1 in E). HPLC detection of the influence of SPINK6 on LTA4H epoxide hydrolase activity Steps: LTA4 methyl ester is activated after freeze-drying (preparation of LTA4: 100...

Embodiment 3

[0038] Immunosuppression experiment

[0039] The 1640 medium containing 100ng / ml PMA stimulated THP-1 to adhere to the wall, overnight, replaced the normal 1640 cell medium, and cultured for 24 hours; starved for 6-12 hours, added SPINK6 to each well and incubated for 5-10 minutes, and the NC group added the same amount of PBS, Add LPS (2μg / ml) and incubate for 6-8h, collect the cell supernatant, use the Elisa method to detect the content of IL-1β, TNF-α, use GraphPadPrism7.0 to draw, and use the unpaired t test to determine the difference between the groups ( figure 2 ). Results: SPINK6 had an inhibitory effect on LPS-induced THP-1 cells to produce inflammatory factors IL-1β and TNF-α, that is, SPINK6 had an immunosuppressive effect.

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Abstract

The invention provides an application of SPINK6 as a marker for detecting AIDS (acquired immune deficiency syndrome) and belongs to the technical field of biomedicine, the SPINK6 can be used for promoting HIV (human immunodeficiency virus) to infect an organism by inhibiting the epoxide hydrolase activity of leukotriene A4 hydrolase, and the SPINK6 can be used as the marker for detecting AIDS.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of SPINK6 as a marker for detecting AIDS. Background technique [0002] Acquired immunodeficiency syndrome is an immunodeficiency disease, which is caused by human immunodeficiency virus (HIV) infection with a high mortality rate and extremely harmful diseases. T cell immune function deficiency is the main feature of this disease . According to the statistics of the World Health Organization in 2017, there are 36.9 million people infected with HIV in the world, and this number is still rising. At present, the diagnostic methods for HIV infection are mainly the detection of viral antigens and antibodies in the body fluids of HIV-infected patients. Antibody detection is especially common. However, in the application of antibody detection, the sensitivity is not high, the detection cost is high, and the HIV virus is highly variable. However, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6893G01N2800/26
Inventor 赖仞唐小芃常佳佳
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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