CN108997503A

CN108997503A - People sDR5-Fc recombination fusion protein and its as preparation treatment genital disease drug in application

Info

Publication number: CN108997503A
Application number: CN201710416730.3A
Authority: CN
Inventors: 万晓春; 陈倩; 张青梅; 夏蒙
Original assignee: Shenzhen Zhongke Amshenn Medicine Co Ltd
Current assignee: Shenzhen Zhongke Amshenn Medicine Co Ltd
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2018-12-14
Anticipated expiration: 2037-06-06
Also published as: CN108997503B; WO2018223764A1

Abstract

The present invention relates to a kind of people sDR5-Fc recombination fusion protein and its as the application in the drug of preparation treatment genital disease.Specifically, the present invention constructs the fusion protein of the solvable segment of people DR5 and the Fc segment of human IgG1, the solvable fragment amino acid sequence of its people DR5 is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID No.3 or SEQ ID No.4, the Fc fragment amino acid sequence of human IgG1 is as shown in SEQ ID NO.5, or has and SEQ ID NO.1, SEQ ID NO.2, SEQ ID No.3,95% or more homology of SEQ ID No.4 or SEQ ID NO.5 and recombination fusion protein with the same function.The invention also discloses application of the people sDR5-Fc recombination fusion protein in the drug that preparation treats or prevents inflammation.Wherein inflammation is selected from vesiculitis, prostatitis, urethritis.Recombination fusion protein stability of the invention is good, and bioactivity is high, and can efficiently and safely be used for the treatment of genital disease.

Description

People sDR5-Fc recombination fusion protein and its as preparation treatment genital disease Application in drug

Technical field

The present invention relates to a kind of fusion proteins, and in particular to a kind of people sDR5-Fc recombination fusion protein and its as preparation Treat the application in the drug of vesiculitis.

Background technique

Seminal vesicle is the organ of store essential substances, there is vascular lamina abundant, due to the organs such as seminal vesicle, prostate and the urinary tract, rectum It is close adjacent, therefore easily lead to infect.The stimulation of Inflammatory substances after infection will lead to seminal vesicle wall oedema and congested, work as patient When sperm drainage, smooth muscle is fiercely shunk, and thin vessels is caused to rupture and may occur in which blood sperm.When ejaculatory duct is spread to by inflammation, will lead It causes seminal vesicle ejaculatory duct ampulla and ejaculation tube opening narrow, the reaction of inflammation is further aggravated, and facilitate the formation of calculus, Opposite calculus has aggravated luminal stenosis again, causes the recurrence and delay of infection.Vesiculitis often occurs with prostatitis simultaneously, causes a disease Bacterium is mostly hemolytic streptococcus, staphylococcus aureus and Escherichia coli etc., is divided into acute and chronic two class of vesiculitis, if anxious Property phase inflammation does not control thoroughly, then is easy to switch to chronic seminal vesiculitis.Cardinal symptom include blood sperm, perineum falling inflation, painful ejaculation very To some patientss with obstructive azoospermia infertility, the course of disease is longer.Vesiculitis will affect the reproductive system of male, can make to suffer from There are the complication such as sterility in person.

The base therapy principle of vesiculitis is: acute stage, is based on antibiotic treatment.Therapeutic agent mainly includes following several Kind:

(1) cefuroxime axetil tablets

Cefuroxime axetil tablets are second generation cephalosporin class antibiotic.It takes orally after gastrointestinal tract absorbs, under esterase effect It is hydrolyzed to cefuroxime rapidly and plays antibacterial action.The mechanism of action of cefuroxime axetil tablets is the conjunction for inhibiting bacteria cell wall At.Common adverse reaction is all gastrointestinal reaction such as nausea,vomiting,diarrhea and loose stools etc..Occasionally there is pseudomembranous enteritis.

(2) erythromycin

Erythromycin is macrolide antibiotics, and antimicrobial spectrum is approximate with penicillin, has stronger inhibition to gram positive bacteria Effect.Mechanism of action is mainly combined with the 50S subunit of ribosome, is inhibited peptidyl transferase, is influenced ribose The shifting process of ribosome interferes peptide chain growth, inhibits the synthesis of bacterio protein, be bacteriostatic agent.1) adverse reaction includes Gastrointestinal reaction: have diarrhea, Nausea and vomiting, gastric colic, dispute pain, stomach receive decline etc., incidence, reaction and dosage size It is related.2) allergic reaction: can there are nettle rash and drug fever.Show as drug fever, fash, eosinophilia etc..3) liver Damage: can cause liver damage, if serum alanine aminotransferase increases, jaundice etc. occur.

(3) quinolone drugs anti-infective therapy

Quinolone antibiotics, for target, and divide bacterial cell no longer with the DNA (DNA) of bacterium.This class The adverse reaction of drug mainly has: 1. gastrointestinal reaction: Nausea and vomiting, discomfort, pain etc.；2. reaction hub: have a headache, is dizzy, Dyssomnia etc., and mental symptom can be caused；3. can be induced insane since this class drug can inhibit the effect of γ-aminobutyric acid Epilepsy has epilepsy medical history person to be used with caution；4. this class drug can influence cartilage development, pregnant woman, skeletal immature children should be used with caution；5. can produce knot Crystalline substance urine, still it is easier to occur in alkaline urine；6. large dosage or prolonged application this class drug easily cause hepatic lesion.

It can be seen that the medicament categories for the treatment of vesiculitis are single at present, offer limited effectiveness needs to develop new therapeutic agent.

Death receptor 5 (Death receptor 5, DR5) is the member of Tumor Necrosis Factor Receptors family, in activation Expression is high in peripheral blood lymphocytes, inflammatory tissue, ischemic tissue and some tumour cells.DR5 receptor and neoplasm necrosis The factor it is apoptosis induction ligand related (Tumor necrosis factor related apoptosis inducing ligand, TRAIL cause a series of signal cascade reaction after) combining, activation caspase-8 causes Apoptosis.

The transmembrane protein that people DR5 albumen is made of 411 amino acid.Wherein 1~55 amino acids are signal peptides, 84~ 179 amino acids be containing 2 rich in cysteine repeat function area chain combined area, 184~206 amino acids be across Film area, intracellular region contain death domain.Soluble DR5 (soluble DR5, sDR5) is DR5 without the solvable of trans-membrane region Property form, due to a lack of trans-membrane region cannot express on cell membrane and it is extracellular to be secreted into.Although sDR5 holding is matched with TRAIL The activity that body combines, but cannot be to intracellular transduction apoptotic signal, the Apoptosis that TRAIL-DR5 can be blocked to mediate reacts. And sDR5 is human body oneself protein, has the advantages that small toxicity, non-immunogenicity, the great critical treatment drug as vesiculitis Potential quality.Currently, sDR5 whether there is therapeutic effect for vesiculitis, report there is no both at home and abroad.

Summary of the invention

Explore and find the emphasis that its effective treatment method is current clinical research.Currently used for treating the medicine of vesiculitis Most of object is antibiotic, and vesiculitis caused by different pathogenic bacteria can only use corresponding antibiotic, and antibiosis is known as very More side effects, liver, Toxicity of Kidney, gastrointestinal side effect, or even will cause abuse of antibiotics.

SDR5-Fc fusion protein is by blocking the DR5 receptor knot of TRAIL and cell membrane surface in conjunction with TRAIL molecule It closes, thus the Apoptosis for blocking TRAIL-DR5 access to induce.In vesiculitis, no matter what pathogenic mechanism is, inevitable final It can be related to the apoptosis of cell.TRAIL-DR5 access participates in vesiculitis caused by many factors, therefore indication range compares antibiotic Greatly.And sDR5-Fc fusion protein derives from the protein component of human body itself, it is more safer than antibiotic.It clinically uses relatively Chinese medicine, the sDR5-Fc fusion protein mechanism of action is clear, and action target spot is special, and toxic side effect is smaller.

One aspect of the invention provides a kind of fusion protein, and the aminoterminal of the fusion protein is human death receptor 5 Solvable segment；C-terminus is the Fc segment of human immunoglobulin(HIg) 1 (IgG1).

In another preferred example, the solvable segment of the human death receptor 5 has such as SEQ ID NO.1, SEQ ID Amino acid sequence shown in NO.2, SEQ ID No.3 or SEQ ID No.4, or have and SEQ ID NO.1, SEQ ID NO.2,95% or more homology of SEQ ID No.3 or SEQ ID No.4 and albumen with the same function.

Preferably, the amino acid sequence of the solvable segment of the human death receptor 5 is as shown in SEQ ID NO.1.

In another preferred example, the Fc segment of the human IgG1 has amino acid sequence shown in SEQ ID NO.5.Compared with Good, the amino acid sequence of the Fc segment of the human IgG1 is as shown in SEQ ID NO.5.

In another preferred example, with connector connection or straight between the solvable segment of human death receptor 5 and the Fc segment of human IgG1 It connects in succession.

Preferably, connector is selected from amino acid sequence, more preferably, connector is selected from 1-100 amino acid sequence or 1-10 Amino acid sequence.

The second aspect of the present invention provides the nucleotide sequence of coding foregoing fusion albumen.

Another aspect of the present invention provides people sDR5-Fc recombination fusion protein above-mentioned and treats or prevents inflammation in preparation Drug in application.

Wherein, inflammation is selected from vesiculitis, prostatitis, urethritis.

Another aspect of the present invention provides a kind of tumor necrosin relative death inducing ligand antagonist and controls in preparation Application in the drug for the treatment of or prevention of inflammation.

Wherein, the tumor necrosin relative death inducing ligand antagonist is selected from people sDR5-Fc above-mentioned recombination and melts Hop protein.

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

Another aspect of the present invention provides people sDR5-Fc recombination fusion protein and treats or prevents blood sperm or seminal vesicle in preparation Application in the drug of swelling.

The present invention discloses above-mentioned recombination fusion protein, can form pharmaceutical preparation together with pharmaceutically acceptable auxiliary material Composition is to more stably play curative effect, the preparations, preferably water such as preparation can commonly be suspended for pharmaceutical field, water needle, freeze-drying Needle or lyophilized preparation can pharmaceutically receive the water needle or lyophilized preparation of above-mentioned recombination fusion protein disclosed by the invention Auxiliary material include one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer, wherein surfactant Including nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)；Triton；Dodecyl sulphur Sour sodium (SDS)；Poloxamer (such as poloxamer 188)；Pluronics；Sodium Laurylsulfate；Myristyl, sub- oil base or ten Eight alkylsarcosines etc., additional amount should make recombination fusion protein granulating trend minimum, and solution stabilizer can be carbohydrate, packet Reducing sugar and nonreducing sugar are included, amino acids include monosodium glutamate or histidine, and alcohols includes propylene glycol, poly- second two The additional amount of one or a combination set of alcohol, trihydroxylic alcohol, advanced sugar alcohol, solution stabilizer should make the preparation eventually formed in this field Technical staff think to reach and keep stable state in stable time, isotonic regulator can be one of sodium chloride, mannitol, Buffer can be one of TRIS, phosphate buffer, histidine buffering liquid.

For sDR5-Fc recombination fusion protein when to animal administration including people, dosage is because of patient in the present invention Age and weight, disease traits and seriousness and administration route and it is different, the result and various feelings of zoopery can be referred to Condition, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 0.01~3000mg/ days.

Above-mentioned recombination fusion protein disclosed by the invention and combinations thereof can also and other vesiculitis, prostatitis, Urethritis therapeutic agent is administered in combination, the treatment for vesiculitis, prostatitis, urethritis.These drugs include classes of anti-infective With anti-inflammatory class.Recombination fusion protein disclosed by the invention and combinations thereof can with above-mentioned classes of anti-infective and anti-inflammatory drug it One or combinations thereof drug combination.

Beneficial effect

1, most of the drug for treating vesiculitis in the prior art is antibiotic and Chinese medicine, and the present invention is the treatment of vesiculitis Provide new thinking.

2, the present inventor sDR5-Fc antibody fusion protein can block the core signal access of Apoptosis, and TRAIL-DR5 withers It dies signal path to be widely present in inflammation caused by many factors, therefore, people's sDR5-Fc antibody fusion protein treats vesiculitis With popularity.

3, people sDR5-Fc antibody fusion protein derives from the albumen of human body itself, and metabolite is amino acid, therefore safety The more other therapeutic agents of property are more excellent.And the mechanism of action is totally different from antibiotic and Chinese medicine, be capable of safety with other medicines Internet of Things are used, and are heightened the effect of a treatment, and rehabilitation is promoted.

Detailed description of the invention

Fig. 1 is amino acid sequence SEQ ID NO.6 and SEQ ID NO.8 recombinant protein Nature comparison figure.

25 hydropathic amino acid analyses are had more in Figure 1A SEQ ID NO.8.Figure 1B-Fig. 1 C SEQ ID NO.6 and SEQ ID NO.8 is respectively compared with human TNF related apoptosis-inducing ligand protein affinity testing result.

Fig. 2 is that sDR5-Fc inhibits mouse vesiculitis to scheme.Fig. 2A-Fig. 2 C physiological saline group mouse seminal vesicle is congested, suffers from Vesiculitis；Fig. 2 D 9mg/kg sDR5-Fc administration group mouse seminal vesicle is normal.

Fig. 3 is that mouse vesiculitis incidence compares figure.It is examined using t, the incidence of physiological saline group mouse vesiculitis is aobvious Work property is higher than sDR5-Fc group, p < 0.05.

Specific embodiment

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

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer Proposed condition.Used various reagents and sample, are commercial product unless otherwise indicated in embodiment.

The design and recombination of the recombination sDR5-Fc expressed sequence of embodiment 1

Inventor by long-term research and many experiments, constructs recombination fusion protein, the expression and pure in Chinese hamster ovary celI Change and obtains albumen.People DR5 soluble fragments are subjected to merging for various ways, mass spectrum point with human immunoglobulin(HIg) IgG1Fc segment Destination protein N-terminal is unstable as the result is shown for analysis, it may appear that multiple amino acid shearings design accordingly and prepare a plurality of amino acid sequence Column, by the Fc segment (SEQ of the solvable segment of the human death receptor 5 of aminoterminal (No.1~4 SEQ ID) and the human IgG1 of c-terminus ID NO.5) composition.Wherein the protein amino acid sequence of SEQ ID NO.1 is most short, by SEQ ID NO.1 and SEQ ID NO.5 shape At recombinant protein contained by splicing isomer ratio it is minimum, be 1% under 37 degree, and SEQ ID NO.2 and SEQ ID NO.5 shape At recombinant protein contained by splicing isomer ratio be 31%；SEQ ID NO.3 is 49%；SEQ ID NO.4 is 52%, aobvious Work property is higher than the splicing isomer ratio of SEQ ID NO.1, therefore the recombination egg that SEQ ID NO.1 and SEQ ID NO.5 is formed White purity is optimal, most stable.And carry out the external Activity determination of recombinant protein (MTT experiment) with L929 cell, as the result is shown by SEQ ID NO.1 and SEQ ID NO.5 formed recombinant protein protein active and SEQ ID NO.3 or SEQ ID NO.4 and The protein active for the recombinant protein that SEQ ID NO.5 is formed does not have difference, obviously higher than SEQ ID NO.2 and SEQ ID The protein active for the recombinant protein that NO.5 is formed.Opposite R&D company sDR5-Fc protein active, SEQ ID NO.2 and SEQ ID NO.5 formed recombinant protein protein active be 126.2%, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and The protein active for the recombinant protein that SEQ ID NO.5 is respectively formed is 300%~400%, SEQ ID NO.1, SEQ ID The recombinant protein and TRAIL molecule that NO.3, SEQ ID NO.4 and SEQ ID NO.5 are respectively formed have very strong binding ability.

SEQ ID NO.1:

SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSP EMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID NO.2:

ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTT RNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEGSSNTKVDKKV；

SEQ ID NO.3:

ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTT RNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID No.4:

AAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGT FREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE；

SEQ ID NO.5:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

In fusion protein of the present invention, between the solvable segment of the DR5 and IgG1Fc segment, it can contain or not Containing catenation sequence.The catenation sequence is usually amino acid sequence or the catenation sequence comprising carboxyl groups, amino acid sequence Length be 1-100 amino acid, the sequence of preferably 1-10 amino acid does not usually have an impact effect to two albumen Sequence.As preferred embodiment of the invention, catenation sequence is not contained between the solvable segment of the DR5 and IgG1Fc segment, As shown in SEQ ID NO.6:

SEQ ID NO.6

SSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSP EMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

The nucleotide sequence of SEQ ID NO.6 fusion protein is encoded as shown in SEQ ID NO.7:

TCCAGCCCCTCAGAGGGATTGTGTCCACCTGGACACCATATCTCAGAAGACGGTAGAGATTGCATCTCCTGCAAATA TGGACAGGACTATAGCACTCACTGGAATGACCTCCTTTTCTGCTTGCGCTGCACCAGGTGTGATTCAGGTGAAGTGG AGCTAAGTCCCTGCACCACGACCAGAAACACAGTGTGTCAGTGCGAAGAAGGCACCTTCCGGGAAGAAGATTCTCCT GAGATGTGCCGGAAGTGCCGCACAGGGTGTCCCAGAGGGATGGTCAAGGTCGGTGATTGTACACCCTGGAGTGACAT CGAATGTGTCCACAAAGAAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAAA

The amino acid sequence provided according to the present invention, those skilled in the art are convenient to that this is made with various known methods The fusion protein of invention.These methods are such as, but not limited to: recombinant DNA method, artificial synthesized, wait [referring to Murray KM, Dahl SLAnn；Pharmacother 1997Nov；31(11):1335-8].

After the amino acid sequence for knowing fusion protein of the invention, those skilled in the art can be convenient according to described Amino acid sequence obtains the gene order for encoding fusion protein of the invention.

As preferred embodiment of the invention, the encoding gene of fusion protein of the invention has shown in SEQ ID NO.7 Sequence expresses fusion protein of the invention particularly suitable for high in eukaryocyte (preferably Chinese hamster ovary celI) using the sequence.

According to nucleotide sequence as described herein, those skilled in the art is convenient to that this hair is made with various known methods Bright code nucleic acid.These methods are such as, but not limited to: PCR, DNA are artificial synthesized etc., and specific method can be found in " molecular cloning Experiment guide ".

For the present invention, any suitable carrier for expression of eukaryon can be used, these carriers can be pcDNA3.1, PDR, pL101 etc..Those skilled in the art can select suitable expression vector according to host cell.As of the invention excellent Mode is selected, when being expressed in eukaryocyte especially Chinese hamster ovary celI (Chinese hamster ovary cell), is expressed using pL101 Carrier.

The present invention provides the host cells for expressing fusion protein of the present invention, wherein containing the volume of fusion protein of the invention Code sequence.The host cell is preferably eukaryocyte, such as, but not limited to Chinese hamster ovary celI, 293 cells, COS cell etc.. As preferred embodiment of the invention, the cell is Chinese hamster ovary celI, can express fusion protein of the invention well, can obtain It obtains and combines activity good, the fusion protein having good stability.

Sequence SEQ ID No.8 ratio SEQ ID No.6 25 amino acid sequences more than the N-terminal, SEQ ID NO.8 sequence is such as Under:

MGVLLTQRTLLSLVLALLFPSMASMSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCT RCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The recombination egg that the recombinant protein that amino acid sequence is SEQ ID NO.6 is SEQ ID NO.8 with respect to amino acid sequence It is white soluble more preferable.Although SEQ ID No.6 only reduces 25 amino acid, inventor in N-terminal relative to SEQ ID No.8 The recombinant protein of SEQ ID NO.6 is surprised to find that by concentration, the high concentration that can achieve 100mg/ml is heavy without generating It forms sediment, is easily formed freeze dried powder and saves steadily in the long term, and high concentration of the recombinant protein of SEQ ID NO.8 in 100mg/ml can produce Raw some visual precipitation is not suitable for using freeze-dried powder dosage form.

In addition, inventors tested a larging the affinity of SEQ ID No.6 and SEQ ID No.8 and trail protein, SEQ ID The affinity ratio SEQ of the recombinant protein of NO.6 and human TNF related apoptosis-inducing ligand albumen (sino biological company, article No. 10409-HNAE) High 1~2 order of magnitude (such as Figure 1B and 1C) of affinity of ID NO.8 and human TNF related apoptosis-inducing ligand albumen.

Embodiment 2: people's sDR5-Fc antibody fusion protein treats vesiculitis.

20 C57BL/6 male mices are divided into 2 groups of (physiological saline group and 9mg/kg fusion protein group SEQ ID NO.6), every group of 10 mouse, every mouse tail vein injection 15mg/kg canavaline (ConA), after 1 hour, every group of mouse point Other tail vein injection gives physiological saline, 9mg/kg fusion protein, and administered volume is 10ml/kg.Continuous injection ConA, physiology Salt water and fusion protein group 15 weeks, then put to death mouse, observe the seminal vesicle of mouse, compared with physiological saline group, 9mg/kg fusion For protein groups mouse seminal vesicle apparently without hyperemia, appearance is normal (see Fig. 2).Therefore, the high-incidence seminal vesicle of physiological saline group mouse conspicuousness The incidence of inflammation, administration group vesiculitis compared with physiological saline group significantly reduces (p < 0.05), is as a result detailed in Fig. 3.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

SEQUENCE LISTING

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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 kind of people sDR5-Fc recombination fusion protein, which is characterized in that the aminoterminal of the fusion protein is human death receptor 5 solvable segments；C-terminus is the Fc segment of human immunoglobulin(HIg) IgG1.

2. people sDR5-Fc recombination fusion protein as described in claim 1, which is characterized in that the human death receptor 5 is solvable Segment has the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID No.3 or SEQ ID No.4, or With with SEQ ID NO.1, SEQ ID NO.2,95% or more homology of SEQ ID No.3 or SEQ ID No.4 and have phase The recombination fusion protein of congenerous.

3. people sDR5-Fc recombination fusion protein as described in claim 1, which is characterized in that the human immunoglobulin(HIg) The Fc segment of IgG1 has the amino acid sequence as shown in SEQ ID NO.5.

4. encoding the nucleotide sequence of the described in any item people sDR5-Fc recombination fusion proteins of claim 1-3.

5. the described in any item people sDR5-Fc recombination fusion proteins of claim 1-3 treat or prevent reproductive system inflammation in preparation Application in the drug of disease.

6. application according to claim 5, wherein genital disease is selected from vesiculitis, prostatitis, urethritis.

7. the medicine that a kind of tumor necrosin relative death inducing ligand antagonist treats or prevents genital disease in preparation Application in object.

8. application according to claim 7, the tumor necrosin relative death inducing ligand antagonist is selected from right It is required that the described in any item people sDR5-Fc recombination fusion proteins of 1-3.

9. application according to claim 7, the genital disease is selected from vesiculitis, prostatitis, urethritis.

10. the described in any item people sDR5-Fc recombination fusion proteins of claim 1-3 treat or prevent blood sperm or seminal vesicle in preparation Application in the drug of swelling.

11. a kind of pharmaceutical composition for treating vesiculitis, it includes the described in any item people sDR5-Fc recombinations of claim 1-3 Fusion protein and its pharmaceutically acceptable auxiliary material.

12. the pharmaceutical composition for the treatment of vesiculitis according to claim 11, wherein also including antibiotic and/or Chinese medicine.