CN111693716A

CN111693716A - Application of SPINK6 as marker for detecting AIDS

Info

Publication number: CN111693716A
Application number: CN202010570568.2A
Authority: CN
Inventors: 赖仞; 唐小芃; 常佳佳
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2020-09-22
Anticipated expiration: 2040-06-19
Also published as: CN111693716B

Abstract

The invention provides an application of SPINK6 in a marker for detecting AIDS, which belongs to the technical field of biomedicine, and SPINK6 can be used as a marker for detecting AIDS by inhibiting the epoxide hydrolase activity of leukotriene A4 hydrolase to promote HIV virus infection on organisms.

Description

Application of SPINK6 as marker for detecting AIDS

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of SPINK6 as a marker for detecting AIDS.

Background

Acquired immunodeficiency syndrome is an immunodeficiency disease, a highly fatal disease with high mortality caused by Human Immunodeficiency Virus (HIV) infection, and T-cell immune function deficiency is a major feature of this disease. According to the data statistics of the world health organization in 2017, 3690 million people in the world are infected with HIV, and the value is still increasing. At present, the diagnosis mode aiming at HIV infection is mainly a method for detecting virus antigens and antibodies in body fluid of HIV infected people, the antibody detection is particularly common, but in the application process of the antibody detection, the reagent for diagnosing and detecting HIV still has the defects and shortcomings due to the characteristics of low sensitivity, high detection cost and high variability of HIV virus. Therefore, the development and exploration of related detection markers have great social demands and important social significance.

The LTB4-BLT signaling pathway is involved in the defense response and inflammatory pathological processes of the body. Immune effector T cells migrate through the LTB4-BLT signaling pathway and participate in inflammation and immune responses in the body. LTB4 is a metabolite of arachidonic acid and has the function of recruiting inflammatory cells and lymphocytes to the site of inflammation, e.g. it activates leukocytes and induces the secretion of some cytokines such as IL-2. LTA4H (leukotriene A4 hydrolase) is a key enzyme catalyzing the production of LTB4, and is an important target for anti-inflammatory drug design. No endogenous inhibitor of LTA4H has been found. The molecular structure of LTA4H shows that the two active pocket sites of epoxide hydrolase and aminopeptidase overlap, so that its inhibitor tends to inhibit both activities simultaneously. When LTA4H is used as a hydrolase, the substrate LTA4 binding pocket binds to LTA4 and is hydrolyzed to generate the inflammatory mediator LTB 4. When LTA4H is used as aminopeptidase, the other pocket can bind chemotactic tripeptide, degrade chemotactic tripeptide, and has inflammation inhibiting effect. LTA4H is thus a zinc-containing bifunctional enzyme with both pro-inflammatory and anti-inflammatory activities. LTB4 has high physiological activity, plays an important role in many inflammatory diseases, immune system diseases, tumorigenesis and allergy, and cell proliferation and differentiation processes, and research on LTB4 is expanded to the aspects of transcription, HIV and immunology. BLT is its specific receptor. LTB4 functions by binding to its receptor. The path is a new path for researching an inflammatory immune generating mechanism, is very useful for immunosuppression, organ transplantation immunosuppression and HIV entry cell inhibition by intervening the LTB4-BLT signal path, and can be used as a new target for treating and intervening diseases such as chronic inflammation and the like.

SPINK6 (SerineProteeImibiatoof Kazal-type6) is a new member of the recently discovered serine protease inhibitor family, and the amino acid sequence of SPINK6 is highly conserved in various organisms and expressed in various important tissues, showing its importance in evolution. The related research at present mainly relates to the occurrence and development of some diseases involved in the inhibition of the activity of KLK (kallikrein) family by SPINK6, and the research on the function of SPINK6 and an inflammatory target LTA4H in disease regulation is not reported.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of SPINK6 as a marker for detecting aids, and SPINK6 can be used as a marker for detecting aids.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an application of SPINK6 as a marker for detecting AIDS.

The invention also provides application of SPINK6 in preparation of medicines for inhibiting epoxide hydrolase activity of leukotriene A4 hydrolase as LTA4H endogenous inhibitor and as inflammation inhibitor and regulating and controlling downstream related inflammatory diseases.

Preferably, the amino acid sequence of the SPINK6 is shown in SEQ ID No. 1.

Preferably, the nucleotide sequence encoding the SPINK6 is shown in SEQ ID No. 2.

The invention provides application of SPINK6 in serving as a marker for detecting AIDS, the expression level of SPINK6 in HIV infected cells is obviously up-regulated, and the inhibition of epoxide hydrolase activity of leukotriene A4 hydrolase promotes HIV virus infection of organisms, so that the SPINK6 can be used as a marker for detecting AIDS.

Drawings

FIG. 1 is a graph of the effect of SPINK6 on LTA4H dual enzyme activity, wherein (A) surface plasmon resonance detects the interaction between SPINK6 and LTA 4H; (B) high pressure liquid chromatography was used to determine the effect of SPINK6 on epoxide hydrolase activity of LTA 4H; (C) statistics of the effect of SPINK6 on epoxide hydrolase activity of LTA4H as measured by high pressure liquid chromatography; (D) enzyme-linked immunosorbent assay detects the effect of SPINK6 on epoxide hydrolase activity of LTA 4H; (E) the effect of SPINK6 on the aminopeptidase activity of the native tripeptide substrate of LTA 4H; (data represent mean ± sd, n-3, p <0.01, n.s. no significant difference);

FIG. 2 shows the results of an immunosuppression experiment in which THP-1 cells were treated with SPINK6, and then inflammatory responses were induced by LPS, and the expression levels of inflammatory factors IL-1 β and TNF- α (n-3, p <0.01) in cell supernatants were measured by ELISA;

FIG. 3 shows plasma SPINK6 levels in HIV patients, wherein plasma from HIV positive and negative volunteers (12 cases each) was subjected to ELISA to detect the expression level of SPINK6 protein (n-12, p < 0.05);

FIG. 4 is a graph showing the expression levels of intracellular SPINK6 following HIV infection, wherein (A) the expression levels of intracellular SPINK6 and LTA4H following HIV-1 infection of CD4T lymphocytes; (B) the intracellular expression level of SPINK6 and LTA4H after HIV-1 infects human H9T lymphocytes; (C) after HOS-CD4-CCR5, Hela-T4 and Tzm-bl cells are infected by two virus subtypes of HIV-1, the expression level of SPINK6 is increased; (NC is not infected by HIV virus, PC is infected group, data represent mean ± standard deviation, n-3,. p < 0.05);

FIG. 5 shows the effect of SPINK6 on the infection rate of HIV virus by pre-treating primary CD4 cells with SPINK6, incubating HIV-1 virus with the cells, and detecting the copy number of the virus in the cells. (NC for uninfected group, PC for infected group, data represent mean ± standard deviation, n-3, p < 0.05).

Detailed Description

The invention provides an application of SPINK6 as a marker for detecting AIDS. In the invention, the SPINK6 promotes HIV virus infection of organisms by inhibiting the epoxide hydrolase activity of leukotriene A4 hydrolase, and can be used as a marker for detecting AIDS.

In the present invention, the SPINK6 has 57 amino acids, molecular weight of about 6063, and two pairs of disulfide bonds in the molecule to maintain stable structure in the molecule. In the invention, the amino acid sequence of SPINK6 is shown as SEQ ID No.1, and specifically comprises the following steps:

QGGQVDCGEFQDPKVYCTRESNPHCGSDGQTYGNKCAFCKAIVKSGGKISLKHPGKC。

in the invention, the nucleotide sequence of the SPINK6 is shown as SEQ ID No.2, and specifically comprises the following steps:

tcagggaggacaggttgactgtggtgagttccaggaccccaaggtctactgcactcgggaatctaacccac actgtggctctgatggccagacatatggcaataaatgtgccttctgtaaggccatagtgaaaagtggtggaaagatta gcctaaagcatcctggaaaatgctga。

the invention also provides application of SPINK6 in preparation of medicines for inhibiting epoxide hydrolase activity of leukotriene A4 hydrolase as LTA4H endogenous inhibitor and as inflammation inhibitor and regulating and controlling downstream related inflammatory diseases. The dosage form of the medicine is not particularly limited, the SPINK6 can be used in a medically acceptable dosage form, and the content of the SPINK6 in the medicine is not particularly limited, and the content of active substances in the conventional medicine can be used. In the invention, the medicine takes SPINK6 as the only active substance, and the preparation method of the medicine is not particularly limited and the medicine can be prepared by adopting a conventional method.

In the invention, the SPINK6 inhibits the expression of IL-1 beta and TNF-alpha, and the SPINK6 has an immunosuppressive effect and is an inflammation inhibiting factor.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Induced expression and activity identification of SPINK6 polypeptide

Optimizing a sequence corresponding to SPINK6 according to an escherichia coli codon, synthesizing a gene, and constructing a PET-32a prokaryotic expression vector, wherein an expression host bacterium is BL 21. An enterokinase cleavage site is set. After the obtained glycerol strain is subjected to plate streak activation, the strain liquid is subjected to small shaking at the temperature of 37 ℃ and the rpm of 160 overnight, and IPTG induction expression is carried out for 3-5h at the temperature of 28 ℃; and (3) expanding and culturing the SPINK6 overnight, collecting bacteria, resuspending and crushing the bacteria. The fusion protein is purified by a nickel column, then the enterokinase enzyme digestion fusion protein is carried out, and the optimal enzyme digestion condition (the enterokinase concentration is set to be 0.5U/ml, the gradient of the added enterokinase is set to be 1U in a reaction system of 50 mu L, the concentration of SPINK6 is 10mg/ml, and the incubation is carried out for 16h at 21 ℃) is selected to carry out the large-scale enzyme digestion of the SPINK6 fusion protein. The protein was further purified by HPLC analysis and separation (solution A: ultrapure water + 0.1% trifluoroacetic acid, solution B: acetonitrile + 0.1% trifluoroacetic acid, HPLC-C4 preparative column for purification was used in the method, and a gradient of solution B was used for the first injection over one minute). As a result: the size of the molecular weight is identified by mass spectrometry, and the in vitro expression is shown to obtain the mature peptide of SPINK 6. Detecting the inhibitory activity of SPINK6 on serine proteases (rhKLK14, Trypsin) (selecting a proper amount of SPINK6, uniformly mixing with the enzymes to be detected, incubating for 5-10min at room temperature, immediately adding a chromogenic substrate, and detecting an enzyme kinetics curve at 405nm by using an enzyme labeling instrument. setting an enzyme kinetics program that the detection duration is 30min, the detection is carried out once every 30s, and the wavelength of the emission light of the chromogenic substrate is 405 nm). As a result: the SPINK6 protein with the inhibitory activity of serine proteases is obtained by in vitro expression.

Example 2

Detection of the Effect of SPINK6 on the binding and Activity of LTA4H

(1) Binding experiments:

surface plasmon resonance detects the interaction of SPINK6 with LTA 4H. LTA4H was coupled to the chip and binding curves (A in FIG. 1) were plotted using GraphPadPrisms 7.0 using a two-fold dilution of mobile phase SPINK6 concentration (800nM, 400nM, 200nM, 100nM, 20. mu.l/min, binding 2min) with the results: there is some interaction between SPINK6 and LTA 4H.

(2) Activity assay:

the effect of SPINK6 on the epoxide hydrolase activity of LTA4H was determined by HPLC and Elisa methods (B-D in FIG. 1), and the effect of SPINK6 on the aminopeptidase activity of LTA4H to hydrolyze its native tripeptide substrate was determined by the indetrione method (E in FIG. 1). Influence of HPLC detection of SPINK6 on LTA4H epoxide hydrolase Activity step: activation of LTA4 methyl ester after lyophilization (preparation of LTA 4: 100. mu.g LTA4 methyl ester by lyophilization, addition of 50. mu.l of a 9: 1 mixture of ice methanol: NaOH (50%), standing at 4 ℃ for 3h, storage at-20 ℃ for further use); the reaction groups are as follows: LTA4 group, LTA4+ LTA4H group, LTA4+ LTA4H + different concentration SPINK6 group; reaction: adding SPINK 6/PBS with equal amount in different concentration gradients into LTA4H, incubating at 37 deg.C for 15-30min, adding LTA4 substrate, incubating at 37 deg.C for 20min, and adding ice methanol to terminate the reaction; RP-HPLC analysis for LTB4 formation: c18 analytical column, mobile phase methanol: water: acetic acid 70: 30: 0.0025, flow rate 1ml/min, detection wavelength 270 nm. Elisa analysis for LTB4 generation: after the reaction was stopped by adding methanol, 500. mu.l of the lyophilizate was taken out and resuspended in a volume of 100. mu.l, and the LTB4 content was determined by the Elisa method. The indetrione method measures the effect of SPINK6 on the aminopeptidase activity of LTA4H to hydrolyze its natural tripeptide substrate. The method comprises the following steps: 1. and (4) incubating. Mixing LTA4H with SPINK6 of different concentrations, incubating at 37 deg.C for 20-25min, adding tripeptide substrate (Pro-Gly-Pro), and reacting at 37 deg.C for 20 min; 2. the volume was diluted 10-fold (final volume is

SPINK6 expression level 200. mu.l after immune cell infection with HIV-1), adding glacial acetic acid and ninhydrin (25mg/ml in acetic acid and 6M phosphoric acid, heated to 70 deg.C for dissolution) in equal volume; 3. heating the reaction mixture at 100 deg.C for 45min, and cooling to room temperature;

4. extracting with 400 μ l toluene, shaking for 30s, standing for a while to transfer all pigments to the toluene layer, detecting the absorbance at 520nm with a microplate reader, mapping with GraphPadprism7.0, and determining the difference between groups by unpaired t-test.

As a result: the concentration of SPINK6 at 0.4. mu.M significantly inhibited the epoxide hydrolase activity of leukotriene A4 hydrolase, whereas the concentration at 10. mu.M had no effect on the aminopeptidase activity of LTA 4H.

Example 3

Immunosuppression assay

Stimulating THP-1 adherence by using a 1640 culture medium containing 100ng/ml PMA, standing overnight, replacing a normal 1640 cell culture medium, and culturing for 24 hours; starvation treatment is carried out for 6-12h, each well is added with SPINK6 and incubated for 5-10min, an equal amount of PBS is added into an NC group, LPS (2 mu g/ml) is added and incubated for 6-8h, cell supernatant is collected, the content of IL-1 beta and TNF-alpha is detected by an Elisa method, GraphPadPrism7.0 is used for mapping, and unpaired t test is adopted for carrying out difference judgment among groups (figure 2). As a result: SPINK6 has inhibitory effect on LPS inducing THP-1 cell to produce inflammatory factor IL-1 beta and TNF-alpha, i.e. SPINK6 has immunosuppressive effect.

Example 4

Plasma from HIV-infected and uninfected persons were collected in 12 cases, and the extreme values were deleted and plotted for statistics. As a result: levels of SPINK6 expression were significantly upregulated in HIV positive plasma (figure 3).

Example 5

Expression level of SPINK6 after HIV-1 infection of related immune cells

Acquisition of samples of treated cells of HIV-1 infected human CD4T lymphocytes (72H infection), human H9T lymphocytes and HIV-1YU2 as well as HIV-1IIIB infected human HOS-CD4-CCR5, HOS-CD4-CXCR4, Hela-T4, THP-1, Tzm-bl, MT-4, C8166. Beta-actin is used as an internal reference for controlling the loading. Data represent mean ± standard deviation and differences between groups were judged by unpaired t-test (NC: uninfected group, PC: infected group, fig. 4). As a result: from the aspect of immunoblotting experiments, it is proved that at the cellular level, HIV-1 infects some immune cells of human body to cause the expression of SPINK6 to be up-regulated. After HIV-1 and CD4T lymphocytes are incubated for 72h, the expression level of SPINK6 is obviously up-regulated before and after HIV-1 infected cells regardless of whether PHA activates CD4T lymphocytes. While there was no difference in the expression level of LTA4H (A in FIG. 4). In the human H9T lymphocyte virus infection experiment, after the incubation of HIV-1 and cells for 4H, 16H and 72H, the expression level of SPINK6 was up-regulated and significantly different from that of HIV-1 and cells incubated for 0H and NC groups, and there was no difference in the expression level of LTA4H (B in fig. 4). In addition, two subtypes of HIV-1 virus are selected to carry out random infection experiments on target cells, and the results show that after HIV-1YU2 and HIV-1IIIB infect HOS-CD4-CCR5, Hela-T4 and Tzm-bl cells, the expression level of SPINK6 before and after infection is obviously different and the expression of SPINK6 is up-regulated when HOS-CD4-CCR5 and Tzm-bl cells infected by the HIV virus are 24h (C in figure 4).

Example 6

Absolute quantitative Real-timePCR detection of HIV-1 copy number

Extracting RNA from target cells infected with HIV virus, diluting the RNA concentration of each group of samples/standards (used for making a standard curve) to the same concentration, preparing PCR reaction solution on ice according to the components in the following table, and setting a negative control without a template. Each group was replicated three times (fig. 5). As a result: after SPINK6 incubation of primary CD4 cells, the number of virus copies in the cells after HIV-1 virus infects human HOS-CD4-CCR5 cells is increased, which indicates that SPINK6 promotes HIV-1 virus to infect HOS-CD4-CCR5 cells by interfering with LTB4-BLT signal paths.

Real-timePCR reaction system (total reaction volume 20. mu.l):

TABLE 1 reaction System

TABLE 2 procedure

The above examples show that SPINK6 can be used as a marker and a therapeutic target for AIDS detection, SPINK6 is used as an endogenous inhibitor of LTA4H, has an inhibitory effect on the activity of LTA4H epoxide hydrolase, presents a concentration-dependent relationship, and has potential biological application value.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Kunming animal research institute of Chinese academy of sciences

Application of <120> SPINK6 as marker for detecting AIDS

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>57

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Gln Gly Gly Gln Val Asp Cys Gly Glu Phe Gln Asp Pro Lys Val Tyr

1 5 10 15

Cys Thr Arg Glu Ser Asn Pro His Cys Gly Ser Asp Gly Gln Thr Tyr

20 25 30

Gly Asn Lys Cys Ala Phe Cys Lys Ala Ile Val Lys Ser Gly Gly Lys

35 40 45

Ile Ser Leu Lys His Pro Gly Lys Cys

50 55

<210>2

<211>175

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tcagggagga caggttgact gtggtgagtt ccaggacccc aaggtctact gcactcggga 60

atctaaccca cactgtggct ctgatggcca gacatatggc aataaatgtg ccttctgtaa 120

ggccatagtg aaaagtggtg gaaagattag cctaaagcat cctggaaaat gctga 175

Claims

The application of SPINK6 in the detection of AIDS marker.
The application of SPINK6 in preparing medicines for inhibiting the epoxide hydrolase activity of leukotriene A4 hydrolase as LTA4H endogenous inhibitor and as inflammation inhibitor and regulating and controlling downstream related inflammatory diseases.
3. The use according to claim 1 or 2, wherein the amino acid sequence of SPINK6 is shown in SEQ id No. 1.
4. The use according to claim 3, wherein the nucleotide sequence encoding SPINK6 is as shown in SEQ ID No. 2.