CN110818787B - Increase Ly6C + Macrophage antigen and application thereof - Google Patents

Abstract

The invention belongs to the technical field of immunology, and relates to a method for increasing Ly6C ⁺ Peptide fragment of toxoplasma gondii derived antigen molecule of macrophage, toxoplasma gondii microwire body protein (Toxoplasma gondii microneme protein, tgMIC 3) and its application. The peptide is named as MIC3, and the sequence is shown as SEQ ID NO.2, and the invention further discloses the preparation of the peptide for increasing Ly6C ⁺ The application of macrophage antigen as immunopotentiator for enhancing macrophage inflammatory response and the application in preparing medicine for treating infectious diseases. The invention provides a wide application prospect of peptide MIC3 in preparing medicines and preparations for treating infectious diseases.

Description

Increase Ly6C + Macrophage antigen and application thereof

Technical Field

The invention belongs to the technical field of immunology, and relates to a method for increasing Ly6C ⁺ Peptide fragment of toxoplasma gondii derived antigen molecule of macrophage, toxoplasma gondii microwire body protein (Toxoplasma gondii microneme protein, tgMIC 3) and its application.

Background

Regarding the treatment of infectious diseases, the traditional treatment methods are mainly chemical drug treatment, such as virus infection treatment using chemical agents such as amantadine; antibiotics and the like are used for bacterial infection (still established. Diagnosis and treatment of infectious diseases [ M. ]. Henan medical university Press, 2002.). Immunotherapeutic agents are now also increasingly being used as emerging therapies for the treatment of infectious diseases, such as the treatment of viruses with biological agents interferon.

Macrophages are distributed in various tissues of a human body, can maintain in vivo immune balance, resist invasion of external pathogens, and play an important role in human immune defense. In the aspect of bacterial infection, during acute infection, macrophages recognize a molecular pattern related to bacterial pathogen through pattern recognition receptors widely distributed on the surfaces of the macrophages, so that the macrophages are activated to M1-type macrophages, a large amount of typical pro-inflammatory cytokines such as TNF-alpha, IL-1, NO and the like are secreted, and an adaptive immune response is activated; in chronic infections, macrophages are present predominantly in the form of M2 polarization in order to prevent severe sepsis due to excessive inflammatory reactions (Murray P J, et al Nature Reviews Immunology,2011,11 (11): 723-737). In the process of various parasite infections, macrophages play different roles through different polarization states, such as early stage of schistosoma japonicum infection, the surface molecules and secretion factors of the parasites can activate the macrophages, up-regulate the expression of M1 type macrophages iNOS, IL-12 and the like, and further induce NK cells and T cells to secrete important pro-inflammatory factors, such as IL-12, IFN-gamma and the like, so that acute inflammation and insect death are caused; while schistosome infection is mainly secreted by M2 type macrophages in late stage to promote fibrosis and collagen formation and induce organism repair (Herbert D R, et al Immunity,2004,20 (5): 623-635.).

Ly6C ⁺ Macrophages play a positive role in activating the immune response of the body, combating pathogen infection, and the like. Ly6C recruited by peripheral to central centers in a model of chronic encephalitis caused by infection of mice with toxoplasma ⁺ The monocytes have remarkable inflammation promoting function, can secrete IL-1 alpha, IL-1 beta, iNOs, TNF-alpha and the like, and exert local insecticidal action (Biswas A, et al journal of Immunology,2015,194 (7):3223.). In addition, toxoplasma gondii is tasty and capable of rapidly recruiting Ly6C to small intestinal villi ⁺ Macrophages, which secrete inflammatory cytokines such as TNF- α, IL-12, etc., form the first line of defense against acute exogenous infections (Dunay IRE, et al Immunity,2008,29 (2): 306-317).

Some drugs have been reported to induce Ly6C ⁺ Macrophages. Such as acetaminophen (N-acetyl-p-aminophenol, APAP), but since excessive use can cause liver damage, the dosage must be strictly controlled for use. Thus, ly6C can be induced ⁺ Among parasite molecules of macrophages, the search for increased Ly6C ⁺ The polypeptide antigen of the macrophage has better feasibility.

The published toxoplasma gondii microwire body protein 3 (Toxoplasma gondii microneme protein, tgMIC 3) gene has the sequence number AJ132530.1 in Genebank. It is known that a peptide fragment (234 aa to 306 aa) of TgMIC3 can react with the serum of a patient infected with toxoplasma, and can be used for diagnosis of toxoplasmosis, and currently, commercial products exist. The trade name is Active toxoplasma MIC3 protein fragment, manufacturer Abcam, cat# ab73745. But with respect to the peptide pair Ly6C ⁺ The effect of macrophages is not reported.

In conclusion, the search for an increase in Ly6C from insect-derived molecules ⁺ The antigen of macrophage and its application in treating infectious diseases has wide foreground.

Disclosure of Invention

The invention aims to provide a method for increasing Ly6C ⁺ Antigen of macrophages.

The invention relates to an increase Ly6C ⁺ The antigen from macrophages, derived from toxoplasma gondii microwire body protein 3, tgMIC3, 37.9kDa, is composed of 359 amino acids.

The invention relates to an increase Ly6C ⁺ The antigen sequence of macrophage is shown in SEQ ID No.2, and not the full length of TgMIC3 sequence in Genebank (SEQ ID No. 56644.1), but only one of them (234 aa-306 aa), comprising 73 amino acids, is designated MIC3 (full-length protein TgM with toxoplasma gondii microwire body protein 3IC 3). The peptide fragment can effectively bind with the antibody of toxoplasma gondii microwire body protein 3 to induce Ly6C ⁺ Macrophages.

The invention discloses a preparation process of peptide fragment MIC3 (SEQ ID NO. 2).

The peptide fragment MIC3 disclosed by the invention mainly induces the high expression of Ly6C through a TLR11/MyD88 signal channel, so that the inflammatory response of an organism is enhanced.

The invention discloses a peptide segment with a sequence shown as SEQ ID NO.2 for preparing and increasing Ly6C ⁺ Use of macrophage antigens.

The invention discloses application of a peptide segment with a sequence shown as SEQ ID NO.2 as an immunopotentiator for enhancing macrophage inflammatory response.

The invention discloses application of a peptide fragment with a sequence shown as SEQ ID NO.2 in preparing a medicament for treating infectious diseases.

The peptide fragment MIC3 of the invention enhances the pro-inflammatory ability of activated macrophages.

The invention has the beneficial effects that the invention discovers an insect-derived antigen molecule capable of up-regulating the expression of the Ly6C of the macrophage, and has wide application prospect in the aspects of preparing medicines and preparations for treating infectious diseases.

Drawings

FIG. 1 peptide fragment MIC3 increases Ly6C ⁺ Flow chart of macrophages.

FIG. 2 peptide fragment MIC3 increases Ly6C ⁺ Statistical plot of macrophage proportion.

FIG. 3 is a statistical plot of the increase in Ly6C average fluorescence intensity of peptide fragment MIC 3.

FIG. 4 peptide fragment MIC3 increased the expression of macrophage inflammatory transcription factor phospho-NF- κ B p 65.

FIG. 5 cellular localization of peptide fragment MIC 3.

FIG. 6 peptide fragment MIC3 increases Ly6C through MyD88 signaling pathway ⁺ Statistical plot of macrophage proportion.

FIG. 7 is a statistical plot of the increase in Ly6C average fluorescence intensity of peptide fragment MIC3 through MyD88 signaling pathway.

FIG. 8TLR11 mRNA interference efficiency.

FIG. 9 peptide fragment MIC3 passageIncreased Ly6C for TLR11 signaling pathway ⁺ Statistical plot of macrophage proportion.

FIG. 10 is a statistical plot of increases in Ly6C mean fluorescence intensity of peptide fragment MIC3 through TLR11 signaling pathway.

FIG. 11 peptide fragment MIC3 inhibits toxoplasma proliferation in macrophages.

Detailed Description

The invention will be described in detail below with reference to the drawings and the detailed description.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

The present invention will be described in further detail with reference to the following specific preparation examples and application examples. It should be understood that: these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the necessary list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise specified, are the same as those indicated at the first occurrence.

The expression vector pET28a (+) mentioned in the examples of the present invention is available from Solarbio (cat. P3110).

Acquisition of total Toxoplasma gondii cDNA: collecting Toxoplasma gondii tachyzoite from Vero cells infected with Toxoplasma gondii, extracting total RNA of Toxoplasma gondii, and performing reverse transcription to obtain total cDNA of Toxoplasma gondii.

Embodiment one: detection of the native expression of peptide fragment MIC 3.

1. Based on the cDNA sequence of TgMIC3 gene (SEQ ID NO: AJ 132530.1) in Genebank, primers were designed in advance: the upstream primer is 5' -AAAGGA TCCAGG ACT GGA TGT CAT GCC TTC 3 '(SEQ ID NO. 3) (containing the cleavage site BamHI), the downstream primer is 5' -AAACTCGAGTCA GTT ATC GCA TTT GCA GGA-3' (SEQ ID NO. 4) (containing the cleavage site XhoI and stop codon TGA) by Shanghai Yingxi Mannich trade Co., ltd (Invitrogen); by passing throughThe PCR method comprises the steps of amplifying a 234 aa-306 aa sequence of TgMIC3 from total cDNA of toxoplasma gondii, separating a target PCR fragment by agarose gel electrophoresis, recovering tapping glue, and sending to Shanghai Yingwei Jiesky trade company (Invitrogen) for sequencing; the sequencing result of the target fragment is shown as SEQ.ID.NO.1, and the target fragment is translated into an amino acid sequence by using biological software Lasergene, wherein the amino acid sequence is shown as SEQ.ID.NO.2, and the sequence is a section of TgMIC3 protein amino acid sequence (the sequence number is CAB 56644.1) in Genebank.

The ORF sequence of the peptide fragment MIC3 is shown as SEQ ID No.1 and is 219bp.

The amino acid sequence of the peptide fragment MIC3 after translation of the ORF sequence is shown as SEQ.ID.NO.2, and has 73 amino acids in total.

2. Plasmid pGEX-6p-1 and the PCR products recovered in the foregoing were subjected to double digestion with BamHI (NEB, #R3136) and XhoI (NEB, #R0146); separating and identifying the enzyme-digested products by agarose gel electrophoresis, and then respectively separating gel to recover double enzyme-digested products of plasmids and PCR fragments; ligation of vector and PCR product was performed by T4 DNA ligase (NEB, # 0202S) to construct recombinant expression vector pGEX-MIC3; transforming E.col iBL competent cells, adding 800 μl of LB culture medium into each tube, pre-culturing at 37deg.C for 1hr, resuscitating bacteria, and expressing resistance genes on plasmids; the culture was spread on LA plates containing ampicillin and cultured upside down at 37℃for 12 to 16 hours, after which transformants were observed. The transformant was inoculated in LB medium containing ampicillin, and after shaking culture at 37℃for 12hr, the plasmid was extracted, verified by double digestion, and further identified by sequencing company. After the identification is successful, the bacterial liquid is subjected to 1:100, adding 0.2mM IPTG when the OD value reaches 0.6-0.8, and inducing expression at 24 ℃ for 8hr; the cells were collected by centrifugation at 7000rpm for 10 min. Separating soluble protein and inclusion bodies by repeated freeze thawing and ultrasonic breaking of thalli, centrifuging at 15000rpm for 40min, and analyzing the expression quantity and distribution condition of the protein by SDS-PAGE electrophoresis; after identification as soluble expression, purification of the target peptide fragment MIC3 is performed by GST fusion tag protein purification resin; after obtaining the purified peptide fragment MIC3, purity was determined by SDS-PAGE electrophoresis.

Embodiment two: peptide fragment MIC3 stimulated mouse macrophages, effect on expression of Ly6C by mouse macrophages.

The peptide fragment MIC3 was endotoxin-removed using AffinityPak Detoxi-Gel Endotoxin Removing Gel (Thermo, 20339). The mouse macrophage cell line RAW264.7 was stimulated with 4 μg/ml peptide fragment MIC3, while the macrophage cell line RAW264.7 was treated for 24 hours with equal concentrations of OVA as a control. The plates were removed, 2ml Staining Buffer cells were added to each well, and the adherent RAW264.7 cells were gently scraped off using a starting Buffer rinse, gently swirled, and transferred to a 15ml centrifuge tube. Centrifuge at 1500rpm for 5min at 4℃and discard the supernatant. Cells were resuspended in 2ml Staining Buffer, centrifuged at 1500rpm for 5min at 4℃and the supernatant discarded. The cells were resuspended at about 100. Mu. l Staining Buffer, 3. Mu.l PE-labeled F4/80 antibody, 1. Mu.l APC-labeled CD11b antibody, 0.5. Mu.l labeled Ly6C antibody were added, thoroughly mixed, and incubated at 4℃for 30min. Each tube was centrifuged at 1500rpm at 2ml Staining Buffer,4 ℃for 5min and the supernatant was discarded. Each tube was added 500. Mu. l Staining Buffer, cells resuspended, transferred to flow tubes and checked on the machine.

As a result, it was observed that Ly6C after peptide fragment MIC3 acted on macrophages ⁺ Macrophage proportion was significantly up-regulated (fig. 1, fig. 2), and mean fluorescence intensity of Ly6C expression was significantly increased (fig. 3).

Embodiment III: peptide fragment MIC3 stimulated mouse macrophages, effect on expression of the mouse macrophage inflammatory transcription factor NF- κB.

The peptide fragment MIC3 was endotoxin-removed using AffinityPak Detoxi-Gel Endotoxin Removing Gel (Thermo, 20339). The macrophage cell line RAW264.7 was stimulated with 4. Mu.g/ml of peptide MIC3, while the macrophage cell line RAW264.7 was treated with equal concentrations of OVA as negative control and LPS as positive control. The plates were removed, and the protein of the cells from each well was extracted and Western blot was used to detect transcription of the inflammatory transcription factor phospho-NF- κ B p65 (Ser 536).

As a result, it was observed that the inflammatory transcription factor phospho-NF-. Kappa. B p65 (Ser 536) was significantly up-regulated after the peptide fragment MIC3 acted on macrophages (FIG. 4).

Embodiment four: cell localization of peptide fragment MIC3 by fluorescent immunoassay

The peptide fragment MIC3 was endotoxin-removed using AffinityPak Detoxi-Gel Endotoxin Removing Gel (Thermo, 20339). Mouse macrophages were affected with 4 μg/ml peptide MIC3 for 4h, and after the cells were approximately 60-70% confluent, the dishes were removed and washed three times with 1ml Staining Buffer. 1ml of 4% paraformaldehyde was fixed and incubated at room temperature for 30min.1ml Staning Buffer three times. DPBS1 was added dropwise: 10 dilutions of normal serum blocking solution, incubated at 37℃for 30min. Sucking the residual liquid d. The PBS diluted primary antibody was added dropwise, and the mixture was protected from light at 4℃overnight. 1ml Staining Buffer three times. 200 μl/well of DPBS diluted secondary antibody was added dropwise, and the mixture was protected from light at 4deg.C for 30min. The starting Buffer was washed three times. DAPI was added dropwise to 200. Mu.l/well, and incubated at 4℃for 15min in the absence of light. The starting Buffer was washed three times. 100 μl/well of the caplet was added dropwise and the staining was observed by laser confocal microscopy. The results show that the peptide fragment MIC3 can either adhere to the cell membrane of mouse macrophages or enter the cell (fig. 5).

Fifth embodiment: effect of peptide fragment MIC3 on the TLR11/MyD88 signaling pathway of mouse macrophages.

MyD88 Joint protein blocking experiment

When the cell growth enters the logarithmic phase, the cell density is regulated, the cell is inoculated in a culture dish, and the cell is subjected to group culture when the growth density reaches 80%. Resuspension to 2.5X10 with complete DMEM medium ⁴ Each of the cells was inoculated at 1 ml/well into 24-well cell culture plates. After the cells were completely adherent, serum-free DMEM medium (1% green streptomycin+99% 1640) was changed, and starved-cultured for 6 hours. The complete DMEM medium was changed, the experimental group was added with MyD88 inhibitor (20. Mu.g/ml), while a solvent control group with the same volume of DMSO added was established. After pre-incubation for 3 hours, adding peptide fragment MIC3 with a final concentration of 4 mug/ml into an experimental group, adding OVA with the same final concentration into a control group, and continuously culturing for 24 hours to collect cell flow detection Ly6C ⁺ Proportion of cells.

The research result shows that the MyD88 blocking agent acts on the macrophage OVA treatment group and can not induce Ly6C ⁺ The proportion of macrophages is significantly increased; the peptide fragment MIC3 acting on mouse macrophage cell line can significantly induce Ly6C in a culture system without MyD88 blocker ⁺ The proportion of macrophages increases; after blocking MyD88 adaptor protein, the peptide fragment MIC3 induces Ly6C ⁺ The proportion of macrophages was significantly reduced (fig. 6); and giant phagocytosisThe mean fluorescence intensity of cell Ly6C was significantly reduced (fig. 7).

TLR11 interference assay

The day before transfection, cells were seeded in 12-well plates, and 1ml of antibiotic-free medium was added to each well to achieve a cell density of 30% -50% at the time of transfection. Mu.l/well Lipo2000 (gently swirled before use), diluted with 100. Mu.l Opti-MEM, gently mixed and incubated at room temperature for 5 minutes. 40nM (2. Mu.l) TLR11 siRNA was diluted with 100. Mu.l Opti-MEM and gently mixed. Lipo2000 and TLR11 siRNA were gently mixed as soon as possible and allowed to stand at room temperature for 20 min to form a (FAM-) siRNA-transfection reagent mixture. Add to wells containing cells and 800. Mu.l of medium and gently shake to mix. At 37 ℃,5% CO ₂ The incubator was incubated for 6 hours, and the medium was replaced with serum-containing DMEM complete medium. The peptide fragment MIC3 was endotoxin-removed using AffinityPak Detoxi-Gel Endotoxin Removing Gel (Thermo, 20339). qPCR was performed to detect TLR11 gene silencing levels after 24h treatment of RAW264.7 macrophage cell lines with 4 μg/ml peptide fragment MIC3 and OVA. After 24h of either OVA or peptide fragment MIC3, TLR11 siRNA interfered with TLR11 expression was reduced by about 60% compared to the unrelated sequence siRNA control (fig. 8).

Furthermore, we examined the induction of Ly6C by TLR11 interfering with peptide fragment MIC3 using flow cytometry ⁺ Effect of macrophage proportion. The results showed that peptide fragment MIC3 stimulated Ly6C after normal mouse macrophages and siCtrl treated mouse macrophages compared to OVA ⁺ Macrophage proportion was significantly increased, while peptide fragment MIC3 stimulated Ly6C induced by macrophages following interference with siTLR11 ⁺ Macrophage proportion was significantly reduced compared to the first two groups (fig. 9). In addition, peptide fragment MIC3 stimulated macrophages after siTLR11 interference, ly6C also showed a decrease in mean fluorescence intensity compared to the first two groups (fig. 10).

Example six: peptide fragment MIC3 inhibits proliferation of worms in macrophages

The peptide fragment MIC3 was endotoxin-removed using AffinityPak Detoxi-Gel Endotoxin Removing Gel (Thermo, 20339). At cell densities up to 30% -50%, the mouse macrophage cell line RAW264.7 was treated with 4 μg/ml peptide MIC3 peptide, while an equal concentration of OVA was used as a control. After 12 hours, tachyzoites of toxoplasma RH strain were added 1:1, cells were harvested after 24 hours, TRIZOL was used to extract total DNA, and qPCR was used to detect expression levels of toxoplasma ITS1 gene and mouse reference gene GAPDH. The ITS1 upstream primer sequence was 5'-CAA GAA GCG TGA TAG TAT CG-3' (SEQ ID NO. 5) and the downstream primer was 5'-CTG AAG AAA CTC CTG GAA ATC-3' (SEQ ID NO. 6). The GAPDH has an upstream primer sequence of 5'-AGG TCG GTG TGA ACG GAT TTG-3' (SEQ ID NO. 7) and a downstream primer sequence of 5'-TGT AGA CCA TGT AGT TGA GGT CA-3' (SEQ ID NO. 8). The relative number of insect charges was calculated by the ratio of the expression level of ITS1 gene and the expression level of GAPDH.

As a result, it was observed that toxoplasma infection of peptide MIC 3-pretreated macrophages had significantly reduced insect load compared to toxoplasma infection of untreated macrophages (fig. 11).

SEQUENCE LISTING

<110> university of Nanjing medical science

<120> an antigen for increasing Ly6C+ macrophages and use thereof

<130>

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 219

<212> DNA

<213> toxoplasma gondii (Toxoplasma gondii)

<400> 1

aggactggat gtcatgcctt cagggagaac tgcagccctg gtagatgtat tgatgacgcc 60

tcgcatgaga atggctacac ctgcgagtgc cccacagggt actcacgtga ggtgacttcc 120

aaggcggagg agtcgtgtgt ggaaggagtc gaagtcacgc tggctgagaa atgcgagaag 180

gaattcggca tcagcgcgtc atcctgcaaa tgcgataac 219

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<212> PRT

<213> toxoplasma gondii (Toxoplasma gondii)

<400> 2

Arg Thr Gly Cys His Ala Phe Arg Glu Asn Cys Ser Pro Gly Arg Cys

1 5 10 15

Ile Asp Asp Ala Ser His Glu Asn Gly Tyr Thr Cys Glu Cys Pro Thr

20 25 30

Gly Tyr Ser Arg Glu Val Thr Ser Lys Ala Glu Glu Ser Cys Val Glu

35 40 45

Gly Val Glu Val Thr Leu Ala Glu Lys Cys Glu Lys Glu Phe Gly Ile

50 55 60

Ser Ala Ser Ser Cys Lys Cys Asp Asn

65 70

<210> 3

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<212> DNA

<213> Artificial sequence (Manual sequence)

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aaaggatcca ggactggatg tcatgccttc 30

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<213> Artificial sequence (Manual sequence)

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aaactcgagt cagttatcgc atttgcagga 30

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<213> Artificial sequence (Manual sequence)

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caagaagcgt gatagtatcg 20

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<212> DNA

<213> Artificial sequence (Manual sequence)

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ctgaagaaac tcctggaaat c 21

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<212> DNA

<213> Artificial sequence (Manual sequence)

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aggtcggtgt gaacggattt g 21

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<212> DNA

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tgtagaccat gtagttgagg tca 23

Claims (1)

1. Peptide fragment with sequence shown as SEQ ID NO.2 in preparation of macrophage increasing Ly6C ⁺ Use of a macrophage fraction reagent.