CN108997503B

CN108997503B - Human sDR5-Fc recombinant fusion protein and application thereof in preparation of medicines for treating inflammation of reproductive system

Info

Publication number: CN108997503B
Application number: CN201710416730.3A
Authority: CN
Inventors: 万晓春; 陈倩; 张青梅; 夏蒙
Original assignee: Shenzhen Amshen Medicine Co ltd
Current assignee: Shenzhen Amshen Medicine Co ltd
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2021-07-23
Anticipated expiration: 2037-06-06
Also published as: WO2018223764A1; CN108997503A

Abstract

The invention relates to a human sDR5-Fc recombinant fusion protein and application thereof in preparing a medicament for treating reproductive system inflammation. Specifically, the invention constructs a fusion protein of a soluble fragment of human DR5 and an Fc fragment of human IgG1, wherein the amino acid sequence of the soluble fragment of human DR5 is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, the amino acid sequence of the Fc fragment of human IgG1 is shown as SEQ ID No.5, or a recombinant fusion protein which has homology with more than SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 595 and has the same function. The invention also discloses application of the human sDR5-Fc recombinant fusion protein in preparing a medicament for treating or preventing inflammation. Wherein the inflammation is selected from seminal vesiculitis, prostatitis, and urethritis. The recombinant fusion protein has good stability and high biological activity, and can be used for efficiently and safely treating reproductive system inflammation.

Description

Human sDR5-Fc recombinant fusion protein and application thereof in preparation of medicines for treating inflammation of reproductive system

Technical Field

The invention relates to a fusion protein, in particular to a human sDR5-Fc recombinant fusion protein and application thereof in preparing a medicament for treating seminal vesiculitis.

Background

The seminal vesicle is an organ for storing sperms and has abundant vascular layers, and the seminal vesicle, the prostate, the urinary tract, the rectum and other organs are closely adjacent, so the infection is easily caused. The irritation of inflammatory substances after infection can cause edema and congestion of the seminal vesicle wall, and when a patient discharges sperm, smooth muscles shrink violently, so that small blood vessels are ruptured and hemospermia can appear. When the ejaculatory duct is spread by inflammation, the abdominal part of the seminal vesicle ejaculatory duct and the opening of the ejaculatory duct are narrowed, the inflammatory reaction is further aggravated, the calculus is formed, and the stenosis of the duct cavity is aggravated by the calculus, so that the infection is relapsed and delayed. Seminal vesiculitis often occurs simultaneously with prostatitis, pathogenic bacteria are mostly hemolytic streptococcus, staphylococcus aureus, escherichia coli and the like, are divided into acute seminal vesiculitis and chronic seminal vesiculitis, and if the acute stage inflammation is not completely controlled, the acute seminal vesiculitis is easy to be converted into the chronic seminal vesiculitis. The main symptoms comprise hemospermia, perineum bulge, ejaculation pain and even partial patients with obstructive azoospermia infertility, and the course of the disease is long. Seminal vesiculitis affects the male reproductive system and can cause complications such as infertility of patients.

The basic therapeutic principles for seminal vesiculitis are: the acute phase is mainly antibiotic treatment. The therapeutic drugs mainly comprise the following medicines:

(1) cefuroxime axetil tablet

The cefuroxime axetil tablet is a second generation cephalosporin antibiotics. After being absorbed by gastrointestinal tract, the cefuroxime sodium can be rapidly hydrolyzed into cefuroxime under the action of esterase to play an antibacterial role. The action mechanism of the cefuroxime axetil tablet is to inhibit the synthesis of bacterial cell walls. The common adverse reactions are gastrointestinal reactions such as nausea, vomiting, diarrhea and loose stool. Occasionally pseudomembranous enteritis.

(2) Erythromycin

Erythromycin is a macrolide antibiotic, has an antibacterial spectrum similar to that of penicillin, and has a strong inhibition effect on gram-positive bacteria. The action mechanism is mainly combined with the 50S subunit of the ribosomes, inhibits peptide acyltransferase, influences the translocation process of the ribosomes, hinders peptide chain growth, inhibits the synthesis of bacterial proteins, and is a bacteriostatic agent. Adverse reactions include 1) gastrointestinal reactions: the symptoms of diarrhea, nausea, vomiting, gastric colic, mouth and tongue pain, anorexia and the like, and the incidence rate and the reaction are related to the dose size. 2) And (3) allergic reaction: there are urticaria and drug-heat. Manifested as drug fever, rash, eosinophilia, etc. 3) Liver damage: can cause liver damage, such as increase of serum alanine aminotransferase, jaundice, etc.

(3) Quinolone medicine anti-infection treatment

Quinolone antibiotics target bacterial deoxyribonucleic acid (DNA) and prevent bacterial cells from dividing. The adverse reactions of the medicines mainly comprise: gastrointestinal tract reaction: nausea, vomiting, malaise, pain, etc.; central reaction: headache, dizziness, poor sleep and the like, and can cause mental symptoms; the medicines can inhibit the function of gamma-aminobutyric acid, so that epilepsy can be induced, and people with epilepsy history can use the medicines with caution; fourthly, the medicines can influence the cartilage development, and the medicines should be used with cautions for pregnant women and children; the urine may produce crystallized urine, which is more likely to occur in alkaline urine; sixthly, liver damage is easy to cause by large dose or long-term application of the medicine.

Therefore, the existing medicines for treating seminal vesiculitis are single in type and limited in curative effect, and new therapeutic medicines are urgently needed to be developed.

Death receptor 5 (DR 5) is a member of the tumor necrosis factor receptor family and is expressed at high levels in activated peripheral blood lymphocytes, inflammatory tissues, ischemic tissues and some tumor cells. The DR5 receptor is combined with Tumor necrosis factor related apoptosis inducing ligand (TRAIL) to induce a series of signal cascade reactions, activating caspase-8 to induce apoptosis.

The human DR5 protein is a transmembrane protein composed of 411 amino acids. Wherein amino acids 1-55 are signal peptides, amino acids 84-179 are chain-like binding regions containing 2 cysteine-rich repeat functional regions, amino acids 184-206 are transmembrane regions, and the intracellular region contains a death domain. Soluble DR5(soluble DR5, sDR5) is a soluble form of DR5 without a transmembrane region, and is secreted outside the cell due to its inability to be expressed on the cell membrane. Although the sDR5 retains the activity of binding with TRAIL ligand, it can not transmit apoptosis signal to the cell, and can block TRAIL-DR 5-mediated apoptosis reaction. And the sDR5 is a human self-protein, has the advantages of low toxicity and no immunogenicity, and has the potential of being used as an important therapeutic drug for seminal vesiculitis. At present, the sDR5 has no report on whether seminal vesiculitis has a treatment effect at home and abroad.

Disclosure of Invention

The search for effective therapeutic methods is the focus of current clinical research. Most of the existing medicines for treating seminal vesiculitis are antibiotics, and seminal vesiculitis caused by different pathogenic bacteria can only adopt corresponding antibiotics, and the antibiotics have many side effects, liver and kidney toxicity, gastrointestinal adverse reactions and even cause abuse of the antibiotics.

The sDR5-Fc fusion protein blocks TRAIL from being combined with DR5 receptor on the surface of cell membrane by being combined with TRAIL molecule, thereby blocking TRAIL-DR5 pathway-induced apoptosis. In seminal vesiculitis, whatever the pathogenic mechanism, apoptosis of cells must eventually be involved. The TRAIL-DR5 pathway is involved in seminal vesiculitis caused by a variety of factors and therefore has a wider range of indications than antibiotics. And the sDR5-Fc fusion protein is derived from the protein component of the human body and is safer than antibiotics. Compared with the traditional Chinese medicine used clinically, the sDR5-Fc fusion protein has definite action mechanism, specific action target and less toxic and side effect.

One aspect of the present invention provides a fusion protein, wherein the amino terminus of the fusion protein is a soluble fragment of human death receptor 5; at the carboxy terminus is the Fc fragment of human immunoglobulin 1(IgG 1).

In another preferred embodiment, said soluble fragment of human death receptor 5 has an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, or a protein having more than 495% homology with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 495 and having the same function.

Preferably, the amino acid sequence of the soluble fragment of the human death receptor 5 is shown in SEQ ID NO. 1.

In another preferred embodiment, the Fc fragment of human IgG1 has the amino acid sequence shown in SEQ ID NO. 5. Preferably, the amino acid sequence of the Fc fragment of human IgG1 is shown in SEQ ID NO. 5.

In another preferred embodiment, the soluble fragment of human death receptor 5 is connected with the Fc fragment of human IgG1 by a linker or directly.

Preferably, the linker is selected from the group consisting of amino acid sequences, more preferably, the linker is selected from the group consisting of 1-100 amino acid sequences, or 1-10 amino acid sequences.

In a second aspect of the invention, there is provided a nucleotide sequence encoding the aforementioned fusion protein.

In still another aspect, the invention provides an application of the human sDR5-Fc recombinant fusion protein in preparation of a medicament for treating or preventing inflammation.

Wherein the inflammation is selected from seminal vesiculitis, prostatitis, and urethritis.

In another aspect, the invention provides the use of an antagonist of TRAIL in the preparation of a medicament for the treatment or prevention of inflammation.

Wherein, the TRAIL antagonist is selected from the human sDR5-Fc recombinant fusion protein.

In another aspect, the invention provides an application of the human sDR5-Fc recombinant fusion protein in preparing a medicament for treating or preventing hematospermia or seminal vesicle swelling.

The invention discloses the above recombinant fusion protein, can form pharmaceutical preparation composition together with pharmaceutically acceptable adjuvants to exert curative effect more stably, the preparation can be suspension, water injection, freeze-drying preparation commonly used in pharmaceutical field, preferably water injection or freeze-drying preparation, for the above recombinant fusion protein water injection or freeze-drying preparation disclosed in the invention, pharmaceutically acceptable adjuvants include one or their combinations of surfactant, solution stabilizer, isotonic regulator and buffer, wherein the surfactant includes nonionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or Tween 80); triton; sodium Dodecyl Sulfate (SDS); poloxamer (such as poloxamer 188); pluronics; sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine, etc., in an amount to minimize the granulation tendency of the recombinant fusion protein, the solution stabilizer may be a saccharide including reducing and non-reducing saccharides, the amino acids include monosodium glutamate or histidine, the alcohols include one of propylene glycol, polyethylene glycol, trihydric alcohol, higher sugar alcohol, or a combination thereof, the solution stabilizer may be added in an amount to maintain the finally formed formulation in a stable state for a time considered to be stable by those skilled in the art, the isotonic adjusting agent may be one of sodium chloride and mannitol, and the buffer may be one of TRIS, phosphate buffer, and histidine buffer.

When the sDR5-Fc recombinant fusion protein of the present invention is administered to animals including humans, the dose to be administered varies depending on the age and body weight of the patient, the nature and severity of the disease, and the route of administration, and it is possible to refer to the results of animal experiments and various cases, and the total dose of administration cannot exceed a certain range. In particular, the dosage of intravenous injection is 0.01-3000 mg/day.

The recombinant fusion protein and the composition thereof disclosed by the invention can also be combined with other therapeutic drugs for seminal vesiculitis, prostatitis and urethritis for administration, and are used for treating seminal vesiculitis, prostatitis and urethritis. These drugs include anti-infective and anti-inflammatory agents. The recombinant fusion protein and the composition thereof disclosed by the invention can be combined with one of the anti-infective drugs and the anti-inflammatory drugs or the combination thereof.

Advantageous effects

1. Most of the medicines for treating seminal vesiculitis in the prior art are antibiotics and traditional Chinese medicines, and the invention provides a new idea for treating seminal vesiculitis.

2. The sDR5-Fc antibody fusion protein of the invention can block a core signal path of apoptosis, and a TRAIL-DR5 apoptosis signal path is widely existed in inflammation caused by various factors, so that the treatment of seminal vesiculitis by the human sDR5-Fc antibody fusion protein has wide application.

3. The human sDR5-Fc antibody fusion protein is derived from human self-protein, and the metabolite is amino acid, so the safety is better than other therapeutic drugs. And the action mechanism is completely different from antibiotics and traditional Chinese medicines, and the traditional Chinese medicine composition can be safely combined with other treatment medicines, so that the curative effect is enhanced, and the rehabilitation is promoted.

Drawings

FIG. 1 is a graph comparing the properties of the recombinant proteins of the amino acid sequences SEQ ID NO.6 and SEQ ID NO. 8.

FIG. 1A shows the hydrophobicity analysis of 25 more amino acids in SEQ ID NO. 8. FIG. 1B-FIG. 1C the results of the affinity detection of SEQ ID NO.6 and SEQ ID NO.8 with human TRAIL protein, respectively.

FIG. 2 is a graph of sDR5-Fc inhibition of mouse seminal vesiculitis. FIG. 2A-FIG. 2C shows seminal vesicle congestion and seminal vesiculitis in mice in saline group; FIG. 2D 9mg/kg sDR5-Fc dosed mice had normal seminal vesicles.

FIG. 3 is a graph comparing the incidence of seminal vesiculitis in mice. Using the t-test, the incidence of seminal vesiculitis was significantly higher in mice in the saline group than in the sDR5-Fc group, with p < 0.05.

Detailed Description

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The reagents and samples used in the examples were all commercially available products unless otherwise indicated.

Example 1 design and recombination of recombinant sDR5-Fc expression sequences

Through long-term research and a large number of experiments, the inventor constructs a recombinant fusion protein, expresses the recombinant fusion protein in CHO cells and purifies the recombinant fusion protein to obtain the protein. The human DR5 soluble fragment and the human immunoglobulin IgG1Fc fragment are fused in various modes, and the mass spectrometry result shows that the N end of the target protein is unstable and can generate a plurality of amino acid shears, so that a plurality of amino acid sequences are designed and prepared, wherein the amino acid sequences consist of the amino-terminal human death receptor 5 soluble fragment (SEQ ID No. 1-4) and the carboxyl-terminal Fc fragment (SEQ ID No.5) of human IgG 1. Wherein the protein amino acid sequence of SEQ ID NO.1 is shortest, the recombinant protein formed by SEQ ID NO.1 and SEQ ID NO.5 contains the lowest proportion of splice isomers, which is 1% at 37 ℃, and the recombinant protein formed by SEQ ID NO.2 and SEQ ID NO.5 contains the splice isomer proportion of 31%; 49% of SEQ ID NO. 3; the content of SEQ ID NO.4 is 52%, and the significance is higher than the proportion of the splice isomers of SEQ ID NO.1, so that the purity of the recombinant protein formed by SEQ ID NO.1 and SEQ ID NO.5 is optimal and most stable. And the results of the in vitro activity detection (MTT test) of the recombinant protein by using the L929 cells show that the protein activity of the recombinant protein formed by SEQ ID NO.1 and SEQ ID NO.5 is not different from that of the recombinant protein formed by SEQ ID NO.3 or SEQ ID NO.4 and SEQ ID NO.5, and is obviously higher than that of the recombinant protein formed by SEQ ID NO.2 and SEQ ID NO. 5. Relative to the activity of sDR5-Fc protein of R & D company, the protein activity of the recombinant protein formed by SEQ ID NO.2 and SEQ ID NO.5 is 126.2%, the protein activity of the recombinant protein formed by SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 is 300-400%, and the recombinant protein formed by SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 and TRAIL molecule have strong binding capacity.

SEQ ID NO.1：

SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID NO.2：

ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEGSSNTKVDKKV；

SEQ ID NO.3：

ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID No.4：

AAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID NO.5：

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

In the fusion protein of the invention, the connection sequence between the soluble fragment of DR5 and the fragment of IgG1Fc may be contained or not contained. The linker sequence is typically an amino acid sequence or a linker sequence comprising an acyl group, the amino acid sequence being 1-100 amino acids in length, preferably 1-10 amino acids in length, typically a sequence that does not affect both proteins. As a preferred mode of the invention, the DR5 soluble fragment and the IgG1Fc fragment do not contain a connecting sequence, and the sequence is shown as SEQ ID NO. 6:

SEQ ID NO.6

SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

the nucleotide sequence of the fusion protein coded by the SEQ ID NO.6 is shown as SEQ ID NO. 7:

TCCAGCCCCTCAGAGGGATTGTGTCCACCTGGACACCATATCTCAGAAGACGGTAGAGATTGCATCTCCTGCAAATATGGACAGGACTATAGCACTCACTGGAATGACCTCCTTTTCTGCTTGCGCTGCACCAGGTGTGATTCAGGTGAAGTGGAGCTAAGTCCCTGCACCACGACCAGAAACACAGTGTGTCAGTGCGAAGAAGGCACCTTCCGGGAAGAAGATTCTCCTGAGATGTGCCGGAAGTGCCGCACAGGGTGTCCCAGAGGGATGGTCAAGGTCGGTGATTGTACACCCTGGAGTGACATCGAATGTGTCCACAAAGAAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAAA

based on the amino acid sequences provided by the present invention, the fusion protein of the present invention can be conveniently prepared by various known methods by those skilled in the art. Such as but not limited to: recombinant DNA methods, artificial synthesis, etc. (see Murray KM, Dahl SLAnn; pharmacother 1997 Nov; 31(11): 1335-8.

After the amino acid sequence of the fusion protein of the present invention is known, the gene sequence encoding the fusion protein of the present invention can be conveniently obtained by those skilled in the art according to the amino acid sequence.

As a preferred mode of the present invention, the encoding gene of the fusion protein of the present invention has a sequence represented by SEQ ID NO.7, and the use of this sequence is particularly suitable for high expression of the fusion protein of the present invention in eukaryotic cells (preferably CHO cells).

The coding nucleic acids of the invention can be readily prepared by one of skill in the art using a variety of known methods based on the nucleotide sequences described herein. Such methods are for example but not limited to: PCR, DNA artificial synthesis, etc., and the specific methods can be found in the molecular cloning laboratory Manual.

For the present invention, any suitable eukaryotic expression vector may be used, and these may be pcDNA3.1, pDR, pL101, etc. One skilled in the art can select an appropriate expression vector depending on the host cell. In a preferred embodiment of the present invention, a pL101 expression vector is used when expression is performed in eukaryotic cells, particularly CHO cells (Chinese hamster ovary cells).

The invention provides host cells expressing the fusion proteins of the invention, which contain the coding sequences for the fusion proteins of the invention. The host cell is preferably a eukaryotic cell, such as but not limited to CHO cells, 293 cells, COS cells, and the like. In a preferred embodiment of the present invention, the cell is a CHO cell which can express the fusion protein of the present invention well and can obtain a fusion protein having a good binding activity and a good stability.

The sequence SEQ ID No.8 has 25 amino acid sequences more at the N end than SEQ ID No.6, and the sequence SEQ ID No.8 is as follows:

MGVLLTQRTLLSLVLALLFPSMASMSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the recombinant protein with the amino acid sequence of SEQ ID NO.6 has better solubility compared with the recombinant protein with the amino acid sequence of SEQ ID NO. 8. Although the amount of the recombinant protein of SEQ ID No.6 is reduced by 25 amino acids at the N-terminus compared with that of SEQ ID No.8, the inventors have surprisingly found that the recombinant protein of SEQ ID No.6 can reach a high concentration of 100mg/ml without precipitation by concentration, and is easily prepared into lyophilized powder for long-term stable storage, while the recombinant protein of SEQ ID No.8 can produce visible precipitation at a high concentration of 100mg/ml, and is not suitable for lyophilized powder.

In addition, the inventors tested the affinity of SEQ ID No.6 and SEQ ID No.8 for TRAIL protein, and the affinity of the recombinant protein of SEQ ID No.6 for human TRAIL protein (sino biological, cat 10409-HNAE,) was 1-2 orders of magnitude higher than the affinity of SEQ ID No.8 for human TRAIL protein (see FIGS. 1B and 1C).

Example 2: the human sDR5-Fc antibody fusion protein treats seminal vesiculitis.

20C 57BL/6 male mice, divided into 2 groups (normal saline group and 9mg/kg fusion protein group SEQ ID NO.6) on average, 10 mice in each group, each mouse injected with 15mg/kg concanavalin (ConA) intravenously at the tail, and after 1 hour, mice in each group were administered with normal saline and 9mg/kg fusion protein by tail vein injection, respectively, in a volume of 10 ml/kg. After the groups of ConA, physiological saline and fusion protein were injected continuously for 15 weeks, the mice were sacrificed and the seminal vesicles of the mice were observed, and the seminal vesicles of the mice in the 9mg/kg fusion protein group were significantly free of hyperemia and normal in appearance, as compared to the physiological saline group (see FIG. 2). Therefore, mice in the saline group had significantly high seminal vesiculitis, and the incidence of seminal vesiculitis was significantly reduced in the administration group compared to the saline group (p <0.05), and the results are shown in fig. 3.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Shenzhen Zhongke Aishen medicine Limited

<120> human sDR5-Fc recombinant fusion protein and application thereof in preparing medicine for treating inflammation of reproductive system

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 109

<212> PRT

<213> fusion protein

<400> 1

Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu

1 5 10 15

Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr

20 25 30

His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser

35 40 45

Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr Val Cys

50 55 60

Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Met Cys

65 70 75 80

Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Gly Asp

85 90 95

Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu

100 105

<210> 2

<211> 138

<212> PRT

<213> fusion protein

<400> 2

Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln

1 5 10 15

Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile

20 25 30

Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr

35 40 45

Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys

50 55 60

Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

65 70 75 80

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu

85 90 95

Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val

100 105 110

Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Gly

115 120 125

Ser Ser Asn Thr Lys Val Asp Lys Lys Val

130 135

<210> 3

<211> 127

<212> PRT

<213> fusion protein

<400> 3

Ile Thr Gln Gln Asp Leu Ala Pro Gln Gln Arg Ala Ala Pro Gln Gln

1 5 10 15

Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile

20 25 30

Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr

35 40 45

Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys

50 55 60

Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr

65 70 75 80

Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu

85 90 95

Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val

100 105 110

Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu

115 120 125

<210> 4

<211> 116

<212> PRT

<213> fusion protein

<400> 4

Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu Cys Pro

1 5 10 15

Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser Cys Lys

20 25 30

Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe Cys Leu

35 40 45

Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro Cys Thr

50 55 60

Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe Arg Glu

65 70 75 80

Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg

85 90 95

Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys

100 105 110

Val His Lys Glu

115

<210> 5

<211> 232

<212> PRT

<213> fusion protein

<400> 5

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 6

<211> 341

<212> PRT

<213> fusion protein

<400> 6

Ser Ser Pro Ser Glu Gly Leu Cys Pro Pro Gly His His Ile Ser Glu

1 5 10 15

Asp Gly Arg Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr

20 25 30

His Trp Asn Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser

35 40 45

Gly Glu Val Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr Val Cys

50 55 60

Gln Cys Glu Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Met Cys

65 70 75 80

Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Gly Asp

85 90 95

Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Glu Pro Lys

100 105 110

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

115 120 125

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

130 135 140

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

145 150 155 160

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

165 170 175

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

180 185 190

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

195 200 205

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

210 215 220

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

225 230 235 240

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

245 250 255

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

260 265 270

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

275 280 285

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

290 295 300

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

305 310 315 320

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

325 330 335

Leu Ser Pro Gly Lys

340

<210> 7

<211> 1023

<212> DNA

<213> Artificial sequence

<400> 7

tccagcccct cagagggatt gtgtccacct ggacaccata tctcagaaga cggtagagat 60

tgcatctcct gcaaatatgg acaggactat agcactcact ggaatgacct ccttttctgc 120

ttgcgctgca ccaggtgtga ttcaggtgaa gtggagctaa gtccctgcac cacgaccaga 180

aacacagtgt gtcagtgcga agaaggcacc ttccgggaag aagattctcc tgagatgtgc 240

cggaagtgcc gcacagggtg tcccagaggg atggtcaagg tcggtgattg tacaccctgg 300

agtgacatcg aatgtgtcca caaagaagag cccaaatctt gtgacaaaac tcacacatgc 360

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 420

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 480

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 540

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 600

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 660

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 720

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 780

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 840

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 900

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 960

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1020

aaa 1023

<210> 8

<211> 366

<212> PRT

<213> fusion protein

<400> 8

Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala

1 5 10 15

Leu Leu Phe Pro Ser Met Ala Ser Met Ser Ser Pro Ser Glu Gly Leu

20 25 30

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

35 40 45

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

50 55 60

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

65 70 75 80

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

85 90 95

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

100 105 110

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

115 120 125

Glu Cys Val His Lys Glu Glu Pro Lys Ser Cys Asp Lys Thr His Thr

130 135 140

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

145 150 155 160

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

165 170 175

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

180 185 190

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

195 200 205

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

210 215 220

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

225 230 235 240

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

245 250 255

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

260 265 270

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

275 280 285

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

290 295 300

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

305 310 315 320

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

325 330 335

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

340 345 350

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

355 360 365

Claims

1. A human sDR5-Fc recombinant fusion protein, wherein the amino terminal of the fusion protein is a human death receptor 5 soluble fragment; the carboxyl terminal is an Fc fragment of human immunoglobulin IgG 1; the human death receptor-5 soluble fragment is an amino acid sequence shown in SEQ ID NO.1, and the Fc fragment of the human immunoglobulin IgG1 is an amino acid sequence shown in SEQ ID NO. 5.

2. A nucleotide molecule encoding the human sDR5-Fc recombinant fusion protein of claim 1.

3. Use of the human sDR5-Fc recombinant fusion protein of claim 1 in the manufacture of a medicament for treating or preventing inflammation of the reproductive system; wherein the inflammation of the reproductive system is selected from seminal vesiculitis.

4. Use of the human sDR5-Fc recombinant fusion protein of claim 1 in the manufacture of a medicament for the treatment or prevention of hemospermia or seminal vesicle swelling.

5. A pharmaceutical composition for treating seminal vesiculitis, which comprises the human sDR5-Fc recombinant fusion protein of claim 1 and pharmaceutically acceptable excipients thereof.

6. The pharmaceutical composition for treating seminal vesiculitis according to claim 5, further comprising antibiotics and/or Chinese herbs.