CN107022557B

CN107022557B - Novel death receptor 5 antibody fusion proteins and uses thereof

Info

Publication number: CN107022557B
Application number: CN201610067931.2A
Authority: CN
Inventors: 万晓春; 李俊鑫; 陈倩
Original assignee: Shenzhen Amshen Medicine Co ltd
Current assignee: Shenzhen Amshen Medicine Co ltd
Priority date: 2016-01-29
Filing date: 2016-01-29
Publication date: 2020-04-21
Anticipated expiration: 2036-01-29
Also published as: CN107022557A

Abstract

The invention relates to a polypeptide with an amino acid sequence shown as SEQ ID NO: 2 (ZJ501-5) and gene SEQ ID NO: 1 and discloses the function of the sDR5-Fc recombinant protein and the gene thereof in preparing the medicine for treating immune liver diseases. In addition, the in vitro bioactivity of the sDR5-Fc protein is 3 times higher than that of an sDR5-Fc product of an R & D systems company, the proportion of N-terminal shearing variants is only about 1%, and the protein drug stability is high.

Description

Novel death receptor 5 antibody fusion proteins and uses thereof

Technical Field

The invention relates to a recombinant protein, in particular to a novel death receptor 5 antibody fusion protein and application thereof.

Background

According to the statistics of the world health organization, the ratio of the hepatitis patients to the healthy population is about 1/12 at the present stage, namely, one hepatitis patient may exist in every 12 persons, and the ratio is nearly ten times of that of the AIDS infected population. At present, nearly 5 hundred million hepatitis patients such as hepatitis B, hepatitis C and the like exist in countries all over the world, and account for 1/12 of the global population. China is the country with the most hepatitis patients, and nearly 30 million people die of liver-related diseases such as cirrhosis and liver cancer every year. More than 50 million people die of primary liver cancer each year worldwide, and up to 80% of primary liver cancer is caused by chronic hepatitis. Of the 4 billion chronic hepatitis patients, 75% live in Asia, while China is the world with the highest burden of hepatitis and liver cancer, and the annual cost for treating hepatitis B in China is up to over 1000 billion.

The occurrence and development of hepatitis and cardiovascular and cerebrovascular diseases have different symptoms but common characteristics, and the key steps of the diseases are related to the rapid apoptosis of liver cells, cardiac muscle cells or brain cells. Apoptosis is an active cell death process caused by the activation of an apoptosis mechanism in a cell by the cell receiving an extracellular death signal, is necessary for maintaining the homeostasis of a human body, and has an important biological effect. However, when cardiovascular and cerebrovascular diseases or hepatitis occur, multiple stressors such as cytokines, oxidative stress, and DNA damage activate apoptotic signals, resulting in the death of a large number of cardiomyocytes, brain cells, or hepatocytes. Apoptosis is the main mode of cell death caused by myocardial infarction, cerebral infarction and hepatitis, is considered as a basic mechanism for the occurrence and development of the diseases, and related molecules of the apoptosis also become key targets for treating the myocardial infarction, the cerebral infarction and the hepatitis. Inhibiting apoptosis can effectively prevent the development of hepatitis or cardiovascular and cerebrovascular diseases.

Death receptor 5 (DR 5) is a member of the Tumor Necrosis Factor (TNF) receptor family, is a specific, high affinity receptor for TNF-related apoptosis-inducing ligand (TRAIL), is low expressed on the surface of normal cells, and is highly expressed in inflammatory, ischemic, or cancerous tissues. Full-length DR5 contains 411 amino acids and belongs to type i transmembrane glycoprotein. After being combined with TRAIL, DR5 can effectively activate intracellular signal transduction pathways to induce the apoptosis of cells. DR5 mediated apoptosis (figure 1) is closely related to myocardial infarction, cerebral infarction and hepatitis induced cell death, and can be used as common drug target of cardiovascular and cerebrovascular diseases and hepatitis

sDR5 (soluble death receptor 5, soluble DR5, abbreviated as sDR5) is a soluble protein, mainly composed of the extracellular domain of DR5, with a size of about 26 kD. The human sDR5 protein competes with DR5 on the surface of a cell membrane to combine TRAIL, thereby blocking or sealing a signal path of the combination of TRAIL and DR5, reducing the apoptosis of functional cells and relieving the damage of the functional cells, and achieving the effects of preventing organ failure and death of patients. In addition, the sDR5 is a human self-protein, has the advantages of small toxicity and no immunogenicity, and has the potential of being used as a 'broad-spectrum' therapeutic drug for diseases such as myocardial infarction, cerebral infarction, hepatitis and the like.

In the prior art, the recombinant human DR5-Fc fusion protein (scientific research reagent) sold by R & D systems company has more amino acids different from other fusion protein drugs on the market at the connection part, and the activity is obviously low; the stability was also poor and different ratios of N-terminal splicing variants were found to be present. These all bring troubles to the later process development and the establishment of quality standards, and are not suitable for biological new drug declaration.

At present, the sDR5-Fc protein serving as a medicine is not available at home and abroad, and belongs to a new medicine. No company can produce sDR5-Fc protein in large scale by GMP (good quality marker: D9563, production is stopped), only 5 sDR5-Fc proteins including Sigma (good quality marker: ALX-522-005), EXBIO antibodies (good quality marker: EXB0007), Abcam (good quality marker: AB83547) and R & D systems (good quality marker: 631-T2) are used as biochemical reagents, the 5 commercial products have different DR5 gene sequences, different engineering cell types are used, the apoptosis inhibiting capability induced by TRAIL protein is poor, the stability is poor, N-terminal shear variants with different proportions exist, and the defects bring troubles to later process development and quality standard establishment.

Disclosure of Invention

Based on the DR5-Fc recombinant protein, the DR5-Fc recombinant protein has high biological activity and good stability.

The invention provides a nucleotide sequence for coding DR5-Fc recombinant protein, which has the following components:

a) SEQ ID NO: 1 in the sequence listing; or

b) And SEQ ID NO: 1; or

c) A sequence which encodes a protein of the same sequence as the nucleotide sequence of a or b but differs from the nucleotide sequence of a or b due to the degeneracy of the genetic code.

The present invention also provides a polypeptide of DR5-Fc recombinant protein encoded by the above-mentioned nucleotide sequence (SEQ ID NO: 1), which has the nucleotide sequence shown in SEQ ID NO: 2, or the amino acid sequence is shown as SEQ ID NO: 2, and the amino acid has one or more substitutions, but the biological activity of the DR5-Fc recombinant protein is not changed.

The invention also aims to provide application of the DR5-Fc recombinant protein and a coding gene sequence thereof.

The specific technical scheme is as follows.

The DR5-Fc recombinant protein is applied to preparing a medicament for treating autoimmune hepatitis.

The nucleotide sequence is applied to preparing the medicine for treating autoimmune hepatitis.

The invention also provides a polypeptide having an amino acid sequence other than SEQ ID NO: 2 belonging to the same sequence and consisting of different basic groups and application thereof.

The specific technical scheme is as follows.

SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. or SEQ ID NO: 6, and the DR5-Fc recombinant protein of the amino acid sequence shown in the specification.

The DR5-Fc recombinant protein is applied to preparing the medicine for treating autoimmune hepatitis.

According to the invention, through long-term research of an inventor, a protein gene sequence is reselected, and the high yield of the sDR5-Fc protein (particularly ZJ501-5) in CHO cells (the primary cell clone screening yield reaches 0.5 g-1 g/L) is realized, the in vitro bioactivity of the sDR5-Fc recombinant protein (particularly ZJ501-5) is 3 times higher than that of an sDR5-Fc product of an R & D system company, the proportion of N-terminal shearing variants is only less than 1%, the protein drug stability is high, and the sDR5-Fc recombinant protein (particularly ZJ501-5) can obviously improve the survival rate of mice with acute autoimmune hepatitis induced by Con A, and has potential application value for treating human autoimmune liver diseases.

Drawings

FIG. 1 is the agarose gel electrophoresis picture of the gene of sDR 5-Fc.

FIG. 2 is a DotBelot assay diagram of DR5-Fc recombinant protein (ZJ 501-5).

FIG. 3 is a non-reducing electrophoresis chart of 8% SDS-PAGE.

FIG. 4 is a SEC-HPLC analysis chart of ZJ501-5 after purification.

FIG. 5 is a TIC map of ZJ501-5 after reduction.

FIG. 6 is a mass spectrum of data from ZJ501-5 after treatment with Biopharmalynx software.

FIG. 7 is a standard curve for the detection of commercial Trail killing activity.

FIG. 8 is a standard curve for biological detection of a commercial DR5-Fc fusion protein.

FIG. 9 is a standard curve for the detection of biological activity of ZJ 501-5.

FIG. 10 is a schematic diagram of the results of liver function tests of ALT and AST in a mouse liver injury model experiment.

FIG. 11 is a schematic diagram of the results of liver function tests of ALT and AST in a mouse liver injury model experiment.

FIG. 12 is a diagram showing the results of HE staining control group in mouse liver injury model experiment.

FIG. 13 is a diagram showing the results of HE staining of sDR5-Fc in a mouse liver injury model experiment.

FIG. 14 is a schematic representation of TUNEL staining of liver tissue sections. Wherein A is the TUNEL staining result of liver tissues of mice in a ConA model group; b is DAPI staining result; c is 9mg/kg sDR5-Fc (501-5) results of TUNEL staining of liver tissue of mice treated, and D is DAPI staining results.

FIG. 15 is a diagram showing the result of TRAIL immunohistochemical staining of liver tissue sections. Wherein, A picture is the result of TRAIL immunohistochemical staining of liver tissues of mice in a ConA model group, which indicates that a large amount of TRAIL positive lymphocytes are recruited to the liver tissues so as to trigger TRAIL-DR 5-mediated liver cell apoptosis; panel B shows the result of TRAIL immunohistochemical staining of liver tissue of mice treated with 9mg/kg sDR5-Fc (501-5), indicating that little apoptosis of liver cells was induced due to the recruitment of few TRAIL-positive lymphocytes to the liver tissue.

FIG. 16 is a graph showing the results of measurement of ALT levels in serum.

FIG. 17 is a graph showing the results of AST level measurement in serum.

FIG. 18 is a graph showing the results of measurement of the level of albumin in serum.

Fig. 19 is a graph showing survival curves of mice.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Design and recombination of recombinant DR5-Fc expression sequence

After long-term experience accumulation, the inventor constructs a fusion protein, carries out fusion of human DR5 and Fc in various ways, and mass spectrometry results show that most target proteins are sheared by 11 amino acids at the N end (ITQQDLAPQQR), and finally screens out a common signal peptide of the fusion protein to remove an intermediate connecting sequence and 18 amino acids at the N end, namely sDR5-Fc (ZJ 501-5). The plasmid was transiently transfected, and the expression supernatant was purified for mass spectrometry and activity analysis.

DNA sequence of ZJ 501-5:

ATGGGTGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCATGtccagcccctcagagggattgtgtccacctggacaccatatctcagaagacggtagagattgcatctcctgcaaatatggacaggactatagcactcactggaatgacctccttttctgcttgcgctgcaccaggtgtgattcaggtgaagtggagctaagtccctgcaccacgaccagaaacacagtgtgtcagtgcgaagaaggcaccttccgggaagaagattctcctgagatgtgccggaagtgccgcacagggtgtcccagagggatggtcaaggtcggtgattgtacaccctggagtgacatcgaatgtgtccacaaagaagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga(SEQ ID NO.1)

amino acid sequence of ZJ501-5

MGVLLTQRTLLSLVLALLFPSMASMSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO.2)

The gene sequence of sDR5-Fc (ZJ501-5) is connected to pL101 eukaryotic expression vector by conventional technical means such as enzyme digestion and connection, and is identified by Hind3 and EcoR1 double enzyme digestion. The agarose gel electrophoresis shows that the target fragment of about 1300bp and the pL101 vector fragment of about 9kb can be seen, the double digestion verification result is shown in figure 1 after the vector is constructed.

Expression and detection of physicochemical properties of di-DR 5-Fc recombinant protein

CHO.K1 cells were transiently transfected with sDR5-Fc/pL101 plasmid using Lipofectamine2000, and 48 hours later, supernatants were collected for DotBlot detection, which gave positive results, as shown in FIG. 2.

Recovery of CHO. K1-S cells at 5X 10⁵Resuspending at 10 ml/ml, followed by FreeStyle^TMCHOExpressionMedium serum-free medium was cultured in the presence of Glutamine added to a final concentration of 8mmol/L, and cultured in a shaking incubator at 37 ℃ under 8% CO2 at 120 rpm. When the number of cells is>1×10⁶Passaging at/ml, adding liquid to 30ml, maintaining cell number 2-5 × 10⁵/ml, for each subsequent passage and maintaining the density to 2-5X 10⁵K1-S, CHO, three passages or more.

One day before transfection, cell density was adjusted to 5-6X 10 after shake flask counting⁵100 ml/ml, cultured in a 37 ℃ 8% CO2 shaking incubator at 120rpm, and transfected the next day.

CHO.K1-S is counted and the activity rate is calculated on the same day of transfection, and the density should be 1.2-1.5X 10⁶Per ml, rate of activity>95 percent. Adjusting the density to 1 × 10⁶30 ml/portion was added to a 100ml shake flask for further use.

Mix FreeStyle by gentle inversion^TMMAX Reagent four times, 37.5ul FreeStyle is taken^TMMAXReagent was added to 0.6ml OPTIpro^TMTo the SFM dilution, 0.6ml of OPTIpro was added simultaneously to 37.5ug of plasmid^TMMixing the two solutions in SFM diluent, and standing at room temperature for 10-20 min.

The incubated mixture was added to the prepared CHO. K1-S cell shake flask, incubated at 37 ℃ for 7 days in 8% CO2 shaking incubator 120rpm, and the product was purified by affinity column and subjected to SDS-PAGE, SEC-HPLC and mass spectrometry.

A mass spectrometry step:

1. the sample was first replaced with buffer solution to 50mM NH4FA (pH6.6) and the concentration was measured.

2. Carrying out enzyme digestion on each sample by using Ides;

3. reduction of disulfide bonds by addition of DTT

4. Mass spectrometry sample injection analysis

See SDS-PAGE (FIG. 3), SEC-HPLC after purification of ZJ501-5 (FIG. 4) (protein purity 99.47%), and TIC after reduction of ZJ501-5 (FIG. 5) and mass spectra of ZJ501-5 data after treatment with Biopharmalynx software (FIG. 6). The ratio of N-terminal cleavages was analyzed according to FIGS. 5 and 6, see the following table, where M (O) indicates that one more methionine was added to the N-terminus of the corresponding protein and oxidized, and S-S indicates that disulfide bonds were formed. O represents the first amino acid oxidized. And in FIG. 5, FIG. 6 is imported into the database and analyzed to obtain the result of subtracting one amino acid from the N-terminal and subtracting two amino acids from the N-terminal.

The ratio of N-terminal shear bodies is shown in the table

The results showed that ZJ501-5 was 99% pure, and only a slight amount of N-terminally cleaved one amino acid and two amino acid variants appeared, in a proportion of 1.11%, with only about 0.11% of the N-terminally cleaved two amino acid variants present.

ZJ501-5 is a target protein with uniform N-terminal obtained by removing an unstable sequence at the N-terminal on the basis of proteins ZJ501-1, ZJ501-2, ZJ501-3 and ZJ 501-4. The N-terminal cleavant ratios of the ZJ501-1, ZJ501-2, ZJ501-3, ZJ501-4, and ZJ501-5 proteins are shown in the following table.

Sample name	Temperature of	N-terminal cutter%
			ZJ501-1	37℃	31.35
ZJ501-2	37℃	34.72
			ZJ501-3	37℃	48.69
ZJ501-4	37℃	51.66
			ZJ501-5	37℃	1.11

As can be seen from the above, the ZJ501-5 protein has the highest drug stability.

The sequences of the proteins ZJ501-1, ZJ501-2, ZJ501-3 and ZJ501-4 are as follows:

ZJ501-1

MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEGSSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.3)

ZJ501-2

MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.4)

ZJ501-3

MGVLLTQRTLLSLVLALLFPSMASMITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.5)

ZJ501-4 amino acid sequence (Signal peptide-target sequence)

MGVLLTQRTLLSLVLALLFPSMASMAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO.6)。

Biological activity identification of three ZJ501-5

1. Detection method for establishing commercialized Trail killing activity

Collecting Jurkat cells in logarithmic growth phase, counting, re-suspending cells with 10% FCS RPMI-1640/DMEM, and adjusting cell density to 8 x 10⁴Perml, 100 ul/well into 96-well cell culture plate, and culturing in 8% carbon dioxide incubator at 37 deg.C for 20-24 hr.

Commercial Trail was resuspended at a final concentration of 500-. The diluted sample was added to a 96-well cell culture plate at 100. mu.l/well and incubated in a 37 ℃ 8% carbon dioxide incubator for 18-22 hours. 20ul of freshly prepared 20:1 mixed MTS/PMS chromogenic solution was added per well, incubation was continued for 3-4 hours at 37 ℃ in an 8% carbon dioxide incubator (without lid), and the A490-A630 values were measured using an MS microplate reader.

Standard curves were fitted using M5 analytical software: the abscissa is the concentration of the sample, the ordinate is A490-A630, A4-parameter equation regression model is selected, and the curve is in an inverse S shape.

1.1 detection result of killing Activity of commercial Trail

Standard curves were fitted using M5 analytical software: the abscissa is the concentration of the sample, the ordinate is A490-A630, A4-parameter equation regression model is selected, and the curve is in an inverse S shape. The software automatically calculated EC50 at 8.382ng/ml and EC90 at 27.65 ng/ml. (see fig. 7).

2. Method for establishing commercial DR5-Fc fusion protein biological activity detection

EC90 for commercial Trail killing activity was calculated by adding Trail concentration to EC90 to the above complete culture medium containing actinomycin D at 0.03ug/ml, and DR5-Fc (R & D systems) was commercialized at a multiple ratio of 10ng/ml and 2 times 15 concentrations using the above complete culture medium containing actinomycin D and EC 90. The diluted sample was added to a 96-well cell culture plate at 100. mu.l/well and cultured in 8% carbon dioxide medium at 37 ℃ for 18-22 hours.

20ul of freshly prepared 20:1 mixed MTS/PMS chromogenic solution was added per well and incubation continued for 3-4 hours at 37 ℃ in an 8% carbon dioxide incubator (without the need for a lid). The A490-A630 value is detected by MS enzyme labeling instrument.

Standard curves were fitted using M5 analytical software: the abscissa is the concentration of the sample, the ordinate is A490-A630, A4-parameter equation regression model is selected, and the curve is in a positive "S" shape. Three different experiments were performed to compare the variation of EC 50.

2.1 measurement of biological Activity of commercial DR5-Fc fusion protein (R & D systems Co.) A standard curve was fitted using M5 analysis software: the abscissa is the concentration of the sample, the ordinate is A490-A630, A4-parameter equation regression model is selected, and the curve is in a positive "S" shape. Three different experiments were performed to compare the variation of EC 50. EC50 was 71.82 ng/ml. See fig. 8.

3ZJ501-5 biological activity and relative activity detection method

As in2, the sample was ZJ501-5(SEQ ID NO.2) and the reference was commercially available DR5-Fc (R & D systems). Experiments were performed in triplicate for two different operators, and the relative biological activity (%) calculated for each experiment ZJ501-5 was commercial DR5-Fc EC 50/sample DR5-Fc EC50 x 100. The variation between different operators and experimental batches was compared.

3.1 biological Activity detection and relative Activity results of ZJ501-5 of the invention

The average of ZJ501-5EC50 is 20.25ng/ml, the commercial DR5-Fc EC50 is 71.82ng/ml, and the relative bioactivity of ZJ501-5 is 355%. (see fig. 9).

The tetrasDR 5-Fc can be used for treating autoimmune hepatitis and viral hepatitis.

The pathologic process of the Con-A induced mouse liver injury model is similar to that of various acute and chronic liver diseases known at present, and particularly the pathologic characteristics of the Con-A induced mouse liver injury model that specific liver injury is induced by activating T lymphocytes can well simulate the pathogenic processes of human autoimmune liver diseases and viral hepatitis.

1.36 Male C57BL/6 mice, 6-8 weeks, were randomly divided into 6 groups of 6 mice each. Model control group, sDR5-Fc treatment group (27mg/kg, 9mg/kg, 3mg/kg and 1mg/kg), positive control group (silibinin27 mg/kg).

2. Each mouse is injected with PBS, sDR5-Fc (invention ZJ501-5)27mg/kg, sDR5-Fc9mg/kg, sDR5-Fc 3mg/kg, sDR5-Fc 1mg/kg and silibinin27mg/kg in the abdominal cavity, and the injection volumes are 20ml/kg

After 3.1 hours, 17mg/kg of Con A was injected into the tail vein of each mouse, the injection volume was 10ml/kg, tail vein blood collection was performed at 8 hours and 24 hours after ConA injection, mice were sacrificed at 48 hours and heart blood collection was performed, livers were collected, fixed with 4% PFA, paraffin-embedded, sectioned, and HE-stained. TUNEL staining of liver tissue sections was performed using the DeadEnd Fluorometric TUNEL System (Promega, G3250) kit. Immunohistochemical staining of liver tissue sections was performed using TRAIL antibody (Santa Cruz, sc-6079). Standing the blood of mouse at room temperature for 1h, centrifuging at 3000rpm and 4 deg.C for 10min, separating upper layer serum, and performing ALT and AST liver function detection with ALT and AST detection kit of Nanjing construction biology company.

See fig. 10-13. FIGS. 10 and 11 show that intraperitoneal injection of 9mg/kg sDR5-Fc can treat ConA-induced acute hepatitis in mice, mainly manifested by a significant decrease in serum transaminase levels due to sDR 5-Fc.

FIGS. 12 and 14 (top row 2) show that after mice were injected with ConA for 48h, extensive hepatocyte death occurred in the liver tissue. FIGS. 13 and 14 (bottom panel 2) show that after mice were injected intraperitoneally with 9mg/kg sDR5-Fc and then subjected to ConA induction for 48h, most of the hepatocytes survived, few apoptotic hepatocytes and intact liver tissue structures. FIG. 15 shows that after intravenous ConA injection in mice, a number of TRAIL-positive lymphocytes were recruited to liver tissue, thereby triggering TRAIL-DR 5-mediated massive apoptosis of hepatocytes; however, after 9mg/kg of sDR5-Fc (501-5) treated mice, few TRAIL-positive lymphocytes were recruited to liver tissues due to sDR5-Fc blocking TRAIL binding to DR5 on the hepatocyte surface, and thus few hepatocyte apoptosis was induced.

And (4) experimental conclusion: the sDR5-Fc can obviously inhibit the rise of serum transaminase in an acute hepatitis mouse model, inhibit pathological changes of the liver and play an obvious liver protection effect. In the dose-dependent experiment, 9mg/kg of sDR5-Fc was injected intraperitoneally as the optimal dose.

4.56 male C57BL/6 mice, 6-8 weeks, randomly divided into 7 groups, each group of 8 blank control group, model administration group (respectively given No. ① - ⑤ gene sequence sDR5-Fc protein, SEQ ID NO.3-SEQ ID NO.6, SEQ ID NO.2, 10 mg/kg).

5. PBS and sDR5-Fc ① - ⑤ 10mg/kg are injected into the tail vein of each mouse, and the injection volume is 10 ml/kg.

After 6.1 hours, injecting ConA 15mg/kg into the tail vein of each mouse with the injection volume of 10ml/kg, collecting blood of submaxillary vein of each mouse 8 hours after ConA injection, standing the blood of the mice at room temperature for 1 hour, centrifuging at 3000rpm and 4 ℃ for 10min, separating the upper serum, and detecting the ALT, AST and albumin levels in the serum by using ALT, AST and albumin detection kits of Nanjing Jiang biological company. See fig. 16, 17 and 18.

And (4) experimental conclusion: compared with the sDR5-Fc protein of other gene sequences, the sDR5-Fc (ZJ501-5) can obviously reduce the transaminase level of the mouse hepatitis induced by ConA (as shown in figures 16 and 17), and under the same dosage, the sDR5-Fc protein of the ZJ501-5 sequence has better curative effect, shows better liver function and can synthesize more serum albumin to be secreted into blood (as shown in figure 18).

7.21 male C57BL/6 mice, 6-8 weeks, were randomly divided into 3 groups, each group consisting of 7 model control group, ② gene sequence sDR5-Fc (ZJ501-2) treatment group 32.2mg/kg, ⑤ gene sequence sDR5-Fc (ZJ501-5) treatment group 21.6 mg/kg. mice were injected into tail vein with PBS, sDR5-Fc ② 32.2mg/kg, sDR5-Fc ⑤ 21.6.6 mg/kg, injection volume was 20 ml/kg.1 hours, mice were injected into tail vein with ConA 15mg/kg, injection volume was 10 ml/kg., survival of mice was observed within 48 hours after Con A injection, and survival curves were drawn, see FIG. 19.

And (4) experimental conclusion: the sDR5-Fc (ZJ501-5) can obviously improve the survival rate of mice with ConA induced acute autoimmune hepatitis, and the curative effect of the sDR5-Fc protein of the ZJ501-5 gene sequence is better than that of the sDR5-Fc protein of the ZJ501-2 gene sequence with the dosage of 32.2mg/kg under the dosage of 21.6 mg/kg.

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.

Claims

1. A nucleic acid molecule encoding a DR5-Fc recombinant protein, having the nucleotide sequence:

a) SEQ ID NO: 1 is shown in the specification; or

b) And SEQ ID NO: 1; or

2. Use of the nucleic acid molecule of claim 1 in the preparation of a medicament for the treatment of autoimmune hepatitis.

DR5-Fc recombinant protein, the amino acid sequence of which is as shown in SEQ ID NO: 2, respectively.

4. Use of the DR5-Fc recombinant protein of claim 3 in the preparation of a medicament for the treatment of autoimmune hepatitis.