CN112125977B - TWEAK-MV1 coupled protein and application thereof in psoriasis resistance - Google Patents

Abstract

The invention discloses a TWEAK-MV1 coupled protein and application thereof in psoriasis resistance, belonging to the technical field of biological medicines. According to the invention, a prokaryotic expression system is adopted, the recombinant TWEAK protein is successfully purified and is successfully coupled with the MV1 protein, so that the TWEAK-MV1 protein capable of inducing keratinocyte apoptosis is prepared, and a new choice is provided for treating psoriasis.

Description

TWEAK-MV1 coupled protein and application thereof in psoriasis resistance

Technical Field

The invention belongs to the field of biological medicines, and relates to a TWEAK-MV1 coupled protein and an application thereof in psoriasis resistance.

Background

Psoriasis is a common chronic inflammatory skin disease, has long course of disease and is easy to relapse. It is currently believed that keratinocyte-centered inflammatory factor release and immunomodulation are critical in the pathogenesis of psoriasis, which results in abnormal keratinocyte cycle processes (active proliferation, inhibited apoptosis). TWEAK, a new member of the TNF family of ligands, exerts biological effects by specifically binding to the unique receptor Fn14 that is distributed on the surface of the target cell membrane. The study reported that the psoriasis skin lesion site Fn14 was significantly highly expressed. The traditional view is that TWEAK/Fn14 signals promote apoptosis and necrosis of normal keratinocytes in vitro. However, the research on keratinocytes in different inflammatory microenvironments shows that TWEAK stimulation has the function of bidirectional regulation of cell fate. The multiple cytokines are utilized to simulate an in vitro psoriasis inflammation microenvironment, can also promote high expression of Fn14, and induce keratinocyte proliferation rather than apoptosis after being applied with TWEAK stimulation.

TNF-inducible regulates cell cycle changes by binding to two subclass receptors, TNFR1 and TNFR 2. Wherein TNF-binding to TNFR1 results in activation of the caspase-8 pathway, leading to apoptosis; TNFR2 can antagonize caspase activating effect caused by TNFR1, and promote NF-response, signal response and cell proliferation. TNFR1 and TNFR2 can be expressed in keratinocytes, vascular endothelial cells, etc., but have a biased distribution that may result in different response effects to TNF-gamma stimulation. TWEAK also promotes keratinocyte to produce anticancer proteins such as C-myc, cIAP2, cflp and the like under psoriasis inflammation, and promotes cells to present a TNF receptor distribution characteristic mainly based on TNFR 2. Thus, the inflammatory microenvironment affects the polarity direction of TWEAK/Fn14 signaling by altering TNFR expression profiles, which in turn regulates keratinocyte proliferation and the psoriatic inflammatory response.

cIAP1 ubiquitinate receptor-interacting serine/threonine protein kinase 1(RIP1) in the cytoplasm, and deletion of cIAP1 in the epidermis leads to keratinocyte death. In keratinocytes overexpressing TNFR2, TWEAK induced more cIAP1 distribution into the cytoplasm, and the cytoplasmic/nuclear ratio of cIAP1 increased with TWEAK stimulation in these cells. However, this effect was abolished by transfection with Fn14 siRNA. Binding affinities between Fn14, TRAF2, cIAP1 and TNFR1 (or TNFR2) molecules were determined by surface plasmon resonance, and we found that Fn14 specifically binds to TRAF2, which further has binding affinity for TNFR1 and TNFR 2; TRAF2 can specifically bind to cIAP1 to form TRAF2-cIAP1 complex. Thus, TRAF2 is a key molecule for forming the Fn14/TRAF2/cIAP1/TNFR complex, and is a key node for mediating TWEAK in promoting the proliferation of psoriasis keratinocytes.

In view of the key role of the TWEAK/Fn14 pathway in the pathogenesis of psoriasis, it would be of great significance to the treatment of psoriasis if a targeted anti-Fn 14 mab/TWEAK-toxin conjugate capable of specifically recognizing Fn14 was designed.

Disclosure of Invention

The invention aims to provide a TWEAK-MV1 coupled protein and an application thereof in resisting psoriasis.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a TWEAK-MV1 coupled protein, wherein the amino acid sequence of the TWEAK-MV1 coupled protein is shown in SED.ID.NO.1.

Further, the nucleotide sequence encoding the TWEAK-MV1 coupled protein is shown in sed.id.no. 2.

The invention also discloses application of the TWEAK-MV1 coupled protein in preparing a medicament for treating psoriasis.

Preferably, the drug is a drug that induces apoptosis of keratinocytes.

The invention also discloses a medicament for treating psoriasis, which is prepared by adding pharmaceutically acceptable pharmaceutic adjuvants into the TWEAK-MV1 coupled protein.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, a prokaryotic expression system is adopted to successfully purify the recombinant TWEAK protein, and the recombinant TWEAK protein is successfully coupled with the MV1 protein to prepare the TWEAK-MV1 protein capable of inducing keratinocyte apoptosis, wherein the MV1 is a specific cIAP1 antagonist, and can form stable connection with biotin through an OH → NH substitution mode and widely exert antagonistic effect. MV1 can promote auto-ubiquitination by inducing conformational changes in cIAP1, ultimately exerting the TRAF2/cIAP1 signal inhibitory effect. And the specific binding of TWEAK and Fn14 enables the TWEAK-MV1 coupler to specifically recognize target cells such as keratinocytes with high expression of Fn14, selectively blocks TRAF2/cIAP cascade signals, weakens the proliferation promoting effect of the TRAF2/cIAP cascade signals on the keratinocytes and leads to the death of the target cells, and the invention can provide a new choice for treating psoriasis.

Drawings

FIG. 1 is a diagram showing a structure of a recombinant plasmid of the present invention;

FIG. 2 is a diagram of carrier information according to the present invention;

FIG. 3 is an enzymatic cleavage map of a recombinant plasmid of the present invention;

FIG. 4 is an SDS-PAGE analysis of a fusion protein expression panel of the present invention;

FIG. 5 is a SDS-PAGE analysis of nickel agarose affinity chromatography purification of the fusion protein of the invention;

FIG. 6 is a SDS-PAGE analysis of the final purified protein;

FIG. 7 shows the results of TWEAK protein concentration measurement;

FIG. 8 is a time-of-flight mass spectrum of TWEAK protein;

FIG. 9 is a time-of-flight mass spectrum of TWEAK-MV 1;

FIG. 10 is a diagram showing the result of HPLC detection of MV1 small molecules;

FIG. 11 is a graph showing HPLC detection results of TWEAK protein;

FIG. 12 is a graph showing the results of HPLC measurements after coupling TWEAK with MV 1;

FIG. 13 is a result of TWEAK-MV1 promoting keratinocyte apoptosis in a psoriasis microenvironment; wherein, A picture shows that soluble tweak (stweak) is successfully constructed by adopting the plasmid; panel B shows TWEAK-MV1 incubated with Fn14 positive keratinocytes; panel C shows that both were applied to Fn14 positive keratinocytes and only TWEAK-MV1 significantly induced apoptosis.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the project carries out theoretical evaluation on the hydrophilicity and hydrophobicity, the signal peptide, the transmembrane domain, the basic structure and the like of the TWEAK protein sequence, designs a plasmid construction scheme according to an evaluation result, constructs a plasmid in a whole-gene synthesis mode, subclones the plasmid on a pET28a expression vector, further transforms the plasmid into Rosetta (DE3) escherichia coli competent cells, cultures, induces and expresses, collects thalli, purifies protein, finally obtains 10mg of recombinant TWEAK protein with the purity of more than 90 percent through verification, and couples MV1 with the TWEAK protein.

1. Material preparation

TABLE 1

2. Prokaryotic expression and purification of TWEAK protein:

2.1 plasmid construction and validation

Amplifying a target gene fragment: designing primers according to the gene sequence and the construction scheme, and amplifying a sufficient amount of PCR products by PCR.

Enzyme digestion: and (3) respectively carrying out enzyme digestion on the PCR product and the plasmid vector according to the enzyme digestion sites designed by the primers.

Connecting target fragments and a vector: and connecting the PCR product after enzyme digestion with a plasmid vector by using ligase.

Obtaining a recombinant plasmid: transferring the connecting liquid into TOP10 competence, detecting and screening positive clones for sequencing verification.

2.2 protein expression

And (3) transformation: transferring the recombinant plasmid into an escherichia coli competent cell, thermally shocking for 90s at 42 ℃, coating the escherichia coli competent cell on a plate containing corresponding kanamycin and chloramphenicol, and culturing;

and (3) activation: selecting a monoclonal to be cultured in a liquid culture medium containing kanamycin and chloramphenicol;

induction: when the OD value reaches 0.6, adding an inducer IPTG (isopropyl-beta-thiogalactoside), continuing to culture, and respectively culturing overnight at 20 ℃ and 37 ℃, wherein the negative control is obtained when the inducer is not added;

and (3) collecting thalli: centrifuging, discarding the supernatant, and collecting the thallus;

and (3) expression detection: adding buffer solution A into the collected thallus for suspension, fully dissolving the thallus by using an ultrasonicator, centrifuging, dissolving the centrifuged precipitate by using buffer solution B, respectively treating the supernatant and the precipitate, preparing a sample, and preparing SDS-PAGE for detection.

Determining the conditional expression: culturing the bacterial liquid in a culture medium containing kanamycin and chloramphenicol, adding 0.5mM inducer IPTG when the OD value reaches 0.6, culturing overnight at 37 ℃ for mass expression, and centrifuging to collect cell thallus.

2.3 protein purification

Collecting crude protein: the cell thallus is dissolved by buffer solution C, and then is subjected to ultrasonic disruption and centrifugation to collect crude supernatant protein.

Balancing: taking 5ml of Ni-NTA, washing the balance column by using a Binding buffer with 5 times of the volume of the column bed, and enabling the flow rate to be 5 ml/min;

and (3) incubation: incubating the crude protein with the balanced column packing for 1 h;

column mounting: putting the incubated product on a column, and collecting and flowing out;

balancing: washing the equilibrium column with a Binding buffer;

impurity washing: washing the column with Washing buffer and collecting the effluent;

and (3) elution: eluting with an Elution buffer, and collecting the effluent;

and (3) purification and detection: and respectively processing the crude protein, the eluate of the washing impurities and the eluate of the elution impurities, preparing samples and preparing SDS-PAGE for detection.

And (3) dialysis concentration: the fraction 5 with better purity is dialyzed into 50mM Tris, 300mM NaCl, 10% Glycerol, 0.1% SKL and pH 7.5, and after the dialysis is finished, the fraction is concentrated by PEG20000, filtered by a 0.22 mu m filter membrane and then subpackaged with 1ml/tube for storage at-80 ℃.

2.4 detection of the protein of interest

SDS-PAGE detection: and (3) treating the purified protein, preparing a sample, running glue, and detecting the molecular weight.

3. TWEAK-MV1 protein coupling:

according to the MV1 structural formula, the tail end OH bond is easily replaced by NH, so that stable connection is formed between the tail end OH bond and a molecule carrying an NH bond end. Previous reports showed that using HATU (C10H15F6N6OP) as an activator, MV1 was combined with diaminohexane (C6H16N2) in Dimethylformamide (DMF) solution and increased four carbon-NH 2 terminal tails; diisopropylethylamine (C8H19N) and 4-AMP (C11H22N2O2) are used as reaction substrates to promote stable connection of MV1 and biotin, and four-carbon-NH is used as a connecting bridge between the two, so that MV1 can be connected to the NH bond end of the TWEAK molecule.

4. Results

4.1 sequence information

4.1.1 optimized Gene sequences

CCATGGGCCATCATCATCATCATCACCGTGCTATCGCAGCACACTACGAGGTCCACCCTCGTCCAGGTCAAGACGGTGCACAGGCTGGTGTAGATGGTACTGTAAGCGGTTGGGAGGAAACCAAGATCAACAGCTCCAGCCCGCTGCGTTACGACCGTCAGATCGGTGAATTCACGGTGATTCGCGCCGGTCTGTACTACCTGTACTGCCAGGTGCACTTCGACGAAGGCAAAGCTGTGTATCTGAAACTGGATCTGCTGGTTAATGGCGTTCTGGCCCTGCGCTGCCTGGAAGAATTCTCCGCGACCGCGGCGTCCTCTCCGGGCCCGCAGCTGCGTCTGTGTCAGGTTTCTGGCCTGCTGCCGCTGCGTCCGGGCTCTTCTCTGCGTATTCGCACTCTGCCGTGGGCTCATCTGAAAGCGGCGCCGTTTCTGACCTATTTTGGCCTGTTCCAGGTTCATTAACTCGAG

4.1.2 Carrier information

The results are shown in FIGS. 1 and 2.

4.1.3 expression of amino acid sequences

The coupled TWEAK-MV1 protein has a length of 153, MW of 17kDa and Predicted pI of 8.50.

The sequence information is as follows:

MGHHHHHHRAIAAHYEVHPRPGQDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQVHFDEGKAVYLKLDLLVNGVLALRCLEEFSATAASSPGPQLRLCQVSGLLPLRPGSSLRIRTLPWAHLKAAPFLTYFGLFQVH

4.2 enzyme digestion detection of recombinant plasmids

The restriction enzyme detection result of the recombinant plasmid is shown in FIG. 3, wherein SM0331(Thermo) is selected by a Marker, and the agarose gel electrophoresis result in FIG. 3 shows that the TWEAK target gene fragment after the PCR product is restricted by ligase is successfully fused with the plasmid to obtain the recombinant plasmid.

4.3 protein expression assay

Protein expression assay results see FIG. 4, in which M represents Protein Marker (Cat. No.: C600525); 1 represents total protein before induction; 2 is the supernatant at 20 ℃; 3, precipitating at 20 ℃; 4 is supernatant at 37 ℃; 5 precipitation at 37 ℃. Through the SDS-PAGE analysis result of the fusion protein expression small test, the obvious TWEAK protein band is shown in the sediment at 37 ℃ after the ultrasonic disruption and centrifugation of the escherichia coli competent cells which are transferred into the recombinant plasmids.

4.4 protein purification assay

The Protein purification and detection results are shown in FIG. 5, in which M is Protein marker (Cat. No.: C600525); 1, loading; 2 is outflow; 3 is 20mM Imidazole elution fraction; 4 is 50mM Imidazole elution fraction; 5-6 is 500mM Imidazole elution fraction; SDS-PAGE was purified by fusion protein nickel agarose affinity chromatography, and a clear TWEAK crude protein band was seen in the eluted fractions.

4.5 fusion destination protein validation

The verification result of the fusion target protein is shown in figure 6, the fusion protein is purified, and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis analysis shows that an obvious band appears at a corresponding position with a theoretical molecular weight of +/-5 kDa, so that the successful purification of the fusion protein can be preliminarily determined.

4.6 concentration determination

The protein concentration is measured to be 0.93mg/ml by using a non-interference protein quantitative kit Cat.No. C503071, the volume of a sample to be measured is 10 mu l, and the results are shown in a graph 7 and the following table 2:

TABLE 2

4.7 bench scale coupling results

The results of the time-of-flight mass spectrometry of the TWEAK protein and the TWEAK-MV1 coupled protein are shown in fig. 8 and 9.

4.8 amplification of the coupling results

The result of the small molecule HPLC assay for MV1 is shown in fig. 10 and table 3 below:

TABLE 3

The HPLC detection results of TWEAK protein are shown in FIG. 11 and Table 4 below

TABLE 4

The results of the HPLC detection of the TWEAK-MV1 coupled protein are shown in FIG. 12 and in Table 5 below:

TABLE 5

5. Effect of TWEAK-MV 1-coupled protein on psoriasis microenvironment cell model

An in vitro psoriasis inflammatory cell model is established by adopting an M5 cytokine combination (comprising IL-1a, IL-17A, IL-22, Oncostatin M and TNF-alpha). Cells were cultured with M5 stimulation to mimic the psoriasis inflammation microenvironment. Cells were cultured in 6-well plates, and near confluency cells were cultured with low 2% fetal bovine serum for 48h, then normal medium was replaced and M5 cytokine (final concentration 10ng/ml, maintenance 2 days) was added. Exogenous TWEAK-MV1, TWEAK or control stimuli were applied. The change of cell cycle and survival rate is detected by experiments such as western blot and fluorescence. Results referring to FIG. 13, panel A in FIG. 13 shows the successful construction of soluble TWEAK (sTWEAK) using plasmids; panel B shows TWEAK-MV1 incubated with Fn14 positive keratinocytes; panel C shows that both were applied to Fn14 positive keratinocytes and only TWEAK-MV1 significantly induced apoptosis.

The results show that: incubating TWEAK-MV1 with the above keratinocytes (Fn14 positive) and TWEAK-MV1 can bind to the cell surface; when the TWEAK and the TWEAK-MV1 are applied to the cells respectively, only the TWEAK-MV1 can obviously induce apoptosis, but the cells of the TWEAK group do not change obviously, which shows that the coupling of the TWEAK and the MV1 can induce the apoptosis of keratinocytes, and a new choice can be provided for treating psoriasis in the future.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

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<120> TWEAK-MV1 coupled protein and application thereof in psoriasis resistance

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cggcgccgtt tctgacctat tttggcctgt tccaggttca ttaactcgag 470

Claims (3)

1. The application of the TWEAK-MV1 coupled protein in preparing the medicine for treating psoriasis is characterized in that the amino acid sequence of the TWEAK protein in the TWEAK-MV1 coupled protein is shown as SED.ID.NO.1, and the nucleotide sequence for coding the TWEAK protein is shown as SED.ID.NO. 2.

2. The use of claim 1, wherein the medicament is a medicament that induces apoptosis of keratinocytes.

3. The medicine for treating psoriasis is characterized by being prepared by adding pharmaceutically acceptable pharmaceutical excipients into TWEAK-MV1 coupled protein, wherein the amino acid sequence of the TWEAK protein in the TWEAK-MV1 coupled protein is shown as SED.ID.NO.1, and the nucleotide sequence for coding the TWEAK protein is shown as SED.ID.NO. 2.

Images