WO2015067113A1 - 人松驰素类似物、其药物组合物及其在医药上的应用 - Google Patents

人松驰素类似物、其药物组合物及其在医药上的应用 Download PDF

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WO2015067113A1
WO2015067113A1 PCT/CN2014/088280 CN2014088280W WO2015067113A1 WO 2015067113 A1 WO2015067113 A1 WO 2015067113A1 CN 2014088280 W CN2014088280 W CN 2014088280W WO 2015067113 A1 WO2015067113 A1 WO 2015067113A1
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Prior art keywords
seq
chain
human relaxin
relaxin
recombinant human
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PCT/CN2014/088280
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English (en)
French (fr)
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张连山
刘佳建
曹国庆
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上海恒瑞医药有限公司
江苏恒瑞医药股份有限公司
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Priority to AU2014346141A priority Critical patent/AU2014346141A1/en
Priority to BR112016009572A priority patent/BR112016009572A2/pt
Priority to EP14860721.1A priority patent/EP3067365A1/en
Priority to KR1020167013109A priority patent/KR20160071472A/ko
Priority to CA2928754A priority patent/CA2928754A1/en
Priority to RU2016117959A priority patent/RU2016117959A/ru
Application filed by 上海恒瑞医药有限公司, 江苏恒瑞医药股份有限公司 filed Critical 上海恒瑞医药有限公司
Priority to US15/033,421 priority patent/US20160326230A1/en
Priority to MX2016005545A priority patent/MX2016005545A/es
Priority to JP2016526060A priority patent/JP2016536306A/ja
Priority to CN201480003657.1A priority patent/CN104870470B/zh
Publication of WO2015067113A1 publication Critical patent/WO2015067113A1/zh
Priority to HK15112223.7A priority patent/HK1211594A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/64—Relaxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2221—Relaxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present disclosure relates to a novel human relaxin analog, a pharmaceutical composition comprising the same, and uses thereof.
  • Relaxin is a polypeptide hormone secreted by the corpus luteum of mammals. Relaxin has a variety of physiological functions in the body, including relaxing the pubic ligament, inhibiting uterine contraction, softening the cervix, stimulating mammary gland development, and affecting milk secretion. In 1926, when studying changes in the pelvic tract during pregnancy, relaxin was first discovered by Frederick Hisaw. Relaxin was thought to be a double-stranded protein in the 1970s and 1980s. The structure of relaxin is similar to that of insulin. Relaxin has been shown to be a member of the family of peptide hormones.
  • the relaxin family is encoded by seven genes: RLN1, RLN2, RLN3 (also known as INSL7), INSL3/RLF, INSL4/EPIL, INSL5/RIF2, and INSL6/RIF1.
  • Most human relaxin in the human circulation is the product of the gene RLN2 encoding.
  • the translation product of RLN2 is a relaxin precursor comprising (from N-terminus to C-terminus): a signal peptide sequence of 24 amino acid residues, a B chain of 29 amino acid residues, and 104-107 amino acid residues A ligated sequence of the base, and an A chain of about 24 amino acid residues.
  • relaxin not only plays an important role in the course of pregnancy, but also affects the vascular structure and function of non-pregnant animals.
  • Relaxin has a wide range of biological effects, including maintaining homeostasis, anti-inflammatory, myocardial protection, dilating blood vessels, and promoting wound healing during mammalian pregnancy and body aging.
  • a particularly important role is anti-fibrosis.
  • the anti-fibrotic effect of relaxin may be an effective measure for future anti-fibrotic treatment.
  • relaxin can be produced by the heart and protects the heart and regulates the extracellular matrix through local receptors. Relaxin has been successfully used to improve cardiac fibrosis in a variety of animal models. Therefore, relaxin is expected to be used in the treatment of human heart fibrotic diseases (Du Xiaojun, Zhou Juan, Baker Heart Institute).
  • a human relaxin (hRelaxin, wild type) precursor is expressed in E. coli; the precursor is folded and renatured after purification; a two-step digestion (using CPB and trypsin, respectively); and further purified to obtain an active Product relaxin.
  • a disadvantage in the prior art is that protein renaturation, two-step digestion and repurification are required in the production process. A multitude of steps leads to a significant reduction in yield and an increase in cost, since each step involves a loss of protein. Moreover, the cost of exogenous enzymes (CPB, trypsin, etc.) itself is high, and the quality of the enzyme also affects the quality and yield of the final product.
  • the present disclosure has carried out the design of novel molecules by changing the amino acid of a specific site of wild-type relaxin, and has discovered a novel human relaxin analog, which is very good. Solve the above problem.
  • the new technical solution shows the following advantages:
  • the amino acid changes at specific sites of relaxin make the structure more suitable for intracellular folding, enzymatic cleavage and secretion, and improve biological activity. This reduces the amount of recombinant protein required per unit dose, thereby reducing costs. At the same time, such an analog makes it easier to develop molecular modifications because of increased activity.
  • the present disclosure yields a specific sequence of human relaxin analogs by design (replacement, deletion, etc. of specific amino acids in the relaxin sequence).
  • the analog is capable of performing protein folding and enzymatic cleavage of a relaxin precursor in a eukaryotic expression system (cell); and when secreted into a fermentation broth, the human relaxin analog of the present disclosure is mature, intact, and Functional relaxin analog.
  • the relaxin analogs obtained by these specific designs have biological activities that are more than 2-fold higher than those of wild-type relaxin.
  • the present disclosure provides a human relaxin analog comprising an A chain and a B chain, the amino acid sequences of the A and B chains being respectively represented by the formula:
  • a chain A 1 LYSALANKCCHVGCTKRSLARFC (24 amino acid residues)
  • a 1 is selected from the group consisting of: Q, D, E, and W;
  • B 14 is selected from the group consisting of: E, D, and N;
  • B 1 and B 2 are simultaneously deleted; when A 1 is Q, B 14 is not E or D.
  • a human relaxin analog as described above, which comprises an A chain and a B chain, the amino acid sequences of the A and B chains being respectively represented by the formula:
  • a chain A 1 LYSALANKCCHVGCTKRSLARFC (24 amino acid residues)
  • a 1 is selected from the group consisting of: Q, D, E, and W;
  • B 14 is selected from the group consisting of: E, D, and N;
  • the A 1 is D, E or W, preferably D.
  • the A 1 is D and the B 14 is D.
  • examples are SEQ ID NO: 134 (A chain) and SEQ ID NO: 135 (B chain), human relaxin analogs formed by disulfide linkage.
  • said B 14 is D.
  • a human relaxin analog as described above wherein the amino acid sequences of the A and B chains are selected from the group consisting of:
  • a human relaxin analog as described above wherein the B chain is linked to the A chain by a linker sequence (abbreviated as L in the present disclosure), the linker sequence having a length of 1 Up to 15 amino acid residues, preferably 2 to 8 amino acid residues, the amino acid sequence of said joining sequence being selected from:
  • a human relaxin analog as described above wherein the N-terminus of the human relaxin analog is linked to a signal peptide sequence (abbreviated as S in the present disclosure), the signal peptide sequence
  • S signal peptide sequence
  • the length is 4 to 15 amino acid residues, preferably 6 to 11 amino acid residues; more preferably, the amino acid sequence of the signal peptide sequence is selected from:
  • an expression precursor for use in the preparation of a human relaxin analog as described above wherein the amino acid sequence of the expression precursor is selected from one of SEQ ID NO: 1 to SEQ ID NO: 26 or A variety.
  • the present disclosure further provides a derivative of a human relaxin analog obtained by PEGylation of a human relaxin analog as described above, in other words, the human relaxin analog as described above is modified with a PEG molecule.
  • the PEG molecule has a molecular weight of from 5 to 100 KDa; in some embodiments, from 10 to 80 KDa; in some embodiments, from 15 to 45 KDa; in some embodiments, from 20 to 40 KDa; In some embodiments, the PEG molecule is branched or linear.
  • the present disclosure further provides a polynucleotide encoding an expression precursor of a human relaxin analog as described above.
  • the polynucleotide is selected from DNA or RNA.
  • the skilled artisan understands that complementary sequences of polynucleotides are also encompassed within the scope of the present disclosure.
  • composition comprising or consisting of:
  • the present disclosure further provides an injectable solution of a human relaxin analog or a derivative thereof, which comprises the pharmaceutical composition as described above in a dissolved form or a soluble form. It will be understood that dry powder or lyophilized powder forms of injectable solutions are also encompassed within the scope of the present disclosure.
  • the present disclosure further provides a human relaxin analog as described above, a human relaxin analog derivative as described above, or a pharmaceutical composition as described above, or an injectable solution as described above, in the preparation for treatment or For use in drugs for preventing fibrotic diseases or cardiovascular diseases, see Cardiovascular effects of relaxin; from basic science to clinical therapy, Nat. Rev. Cardiol. 7, 48-58 (2010); Relaxin reduced renal interstitial fibrosis and slows progression Of renal disease, kindney International, Vol. 59 (2001), pp. 876-882.
  • the present disclosure further provides a method of treating or preventing a fibrotic or cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a human relaxin analog or derivative thereof as described above, or Said pharmaceutical composition.
  • administering When applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, "administering,” “administering,” “treating,” and “treating” refers to an exogenous drug, therapeutic agent, diagnostic agent, or Contact of the composition with an animal, human, subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administering also means treating the cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • treatment refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as administering to a patient a composition comprising any of the binding compounds of the present disclosure, the patient having one or more symptoms of the disease, and the therapeutic agent is known to Symptoms have a therapeutic effect.
  • the therapeutic agent is administered in an amount to effectively alleviate the symptoms of one or more diseases in the patient or population being treated.
  • the amount of effective relief includes the extent to which any such condition is developed by inducing the degradation of such symptoms or inhibiting such symptoms.
  • the amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • embodiments of the present disclosure may have different effects in alleviating the symptoms of the target disease that each patient has, any statistical test method known in the art, such as Student's t-test, chi-square, The test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should statistically significantly reduce the target disease symptoms in the patient.
  • any statistical test method known in the art such as Student's t-test, chi-square, The test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should statistically significantly reduce the target disease symptoms in the patient.
  • the relaxin or human relaxin described in the present disclosure is human relaxin RLN2, which comprises a biologically active full length sequence or partial sequence.
  • Human relaxin contains an A chain and a B chain, and the two are bonded by a disulfide bond.
  • GenBank SEQ ID NO: EAW58770 For the sequence description of human vasopressin used in the present disclosure, see GenBank SEQ ID NO: EAW58770. Human vasopressin 800828 used as a positive control in the present disclosure is wild type.
  • the human relaxin analog precursors described in the present disclosure comprise, in order from the amino terminus to the carboxy terminus, a signal peptide sequence, a B chain or a mutant thereof, a linker sequence, an A chain or a mutant thereof.
  • the length of the signal peptide sequence is 4-15 amino acid residues, preferably 6-11 amino acid residues
  • the length of the B chain or variant thereof is 29 amino acid residues
  • the length of the ligation sequence is 1-15 amino acid residues A base, preferably 2-8 amino acid residues
  • the A chain or variant thereof is about 24 amino acid residues in length.
  • Mutants as referred to in the present disclosure refer to modifications, substitutions, substitutions or substitutions of amino acids in sequence.
  • a preferred mutation of the B chain of the present disclosure is to replace E with D at position 14 (abbreviated as B 14 ) of the B chain, and replace the first position (abbreviated as A 1 )Q of the A chain with D.
  • Ln represents a certain joining sequence (such as L1 to L6) in the present disclosure
  • Sn represents a certain signal peptide sequence (such as S1, S2, S3) in the present disclosure
  • D A1 represents in the A chain.
  • the first amino acid mutation is D
  • D B14 indicates that the 14th amino acid of the B chain is mutated to D
  • Del B1, B2 indicates the deletion of the first and second amino acids of the B chain
  • a (wt) indicating the wild type non-mutated A chain
  • B (wt) represents a wild type non-mutated B chain.
  • Constant modification or “conservative substitution or substitution” means that an amino acid residue in a protein is subjected to other amino acid residues having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) The substitution of the base; even if such amino acid residues in the protein are frequently changed, the biological activity of the protein is not altered. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not alter biological activity (see, eg, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., page 224 Page, 4th edition). In addition, substitution of structurally or functionally similar amino acid residues is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the method of administration, the route, and dosage, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance produced by a source of organism, cell or human.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the expression “transformants” and “transformed cells” includes primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations.
  • the above expression also includes mutant progeny that have the same function or biological activity as the originally transformed cell. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • sequence information from the end or beyond of the target region, thereby enabling the design of oligonucleotide primers; the sequences of these primers are identical or similar to the corresponding strands of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See, for example, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • the PCR used herein is considered to be a nucleic acid polymerization for amplifying nucleic acid test samples.
  • “Pharmaceutical composition” means a mixture containing one or more of the analogs or precursors described herein, as well as other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of other chemical components is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the steps of transforming host cells described in the present disclosure can be carried out using conventional techniques well known to those skilled in the art.
  • the obtained transformant can be cultured by a conventional method, which expresses the polypeptide encoded by the gene of the present disclosure.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • Chemical and/or enzymatic methods for releasing the human relaxin analog from the expression precursor product can be carried out by conventional techniques well known to those skilled in the art, such as trypsin, carboxypeptidase B, lysine endopeptidase C and the like.
  • the codon-optimized DNA sequence of human relaxin 800800 was synthesized by the Overlap PCR method. Six single-stranded DNA fragments were synthesized by Invitrogen as synthetic primers with the following sequence:
  • the synthesis of relaxin was carried out using a KOD plus kit (TOYOBO, Cat. KOD-201) using a two-step PCR.
  • the reaction procedure was 94 ° C for 5 minutes; 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 30 seconds, amplification for 30 cycles; and 68 ° C for 30 minutes to end the PCR amplification procedure.
  • the second step PCR 25 ⁇ L reaction system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, 1 ⁇ L of primers 1 and 6 (10 ⁇ M), 1 ⁇ L of the first round PCR product, 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4 , 15.5 ⁇ L ddH 2 O .
  • the reaction procedure was 94 ° C for 5 minutes; 94 ° C for 30 seconds, 68 ° C for 60 seconds, amplification for 30 cycles; and 68 ° C for 10 minutes to end the PCR amplification procedure.
  • the PCR-synthesized DNA sequence and the pPIC9K expression vector (Invitrogen, Cat. K1750-01) were separately digested with EcoR I/Xho I (New England Biolabs, Cat.R0101S/R0146V), and the obtained fragment was subjected to 1.2% agarose. The gel was recovered, ligated with T4 DNA ligase (New England Biolabs, Cat. M0202V), transformed into DH5 ⁇ competent cells (Tiangen Biochemistry, Cat. CB101-02), and positive clones were selected for sequencing by Invitrogen.
  • the 800 precursor DNA sequence is as follows:
  • 5-10 ⁇ g of the relaxin 800800 expression vector obtained in the above step was linearized with SalI (Takata, Cat. D1080a). Further, 1/10 volume of 3 M sodium acetate and 2 volumes of absolute ethanol were added, mixed well, and placed at -20 ° C for 2 hr. The mixture was centrifuged at high speed (13,000 rpm) for 5 min, the supernatant was removed, and the precipitate was washed twice with 75% ethanol, and the precipitate was inverted and dried, and then dissolved with 10 ⁇ L of ddH 2 O. The linearized plasmid was mixed with 80 ⁇ L of electroporation competent cells (Pichia pastoris GS115, Invitrogen, Cat.
  • G418 G418
  • a single colony was picked from the YPD plate and placed in 4 mL of BMGY medium, and cultured overnight at 30 ° C, 250 rpm. The OD 600 value should be measured the next day and should be between 2-6.
  • the cells were harvested by centrifugation (Beckman Coulter) at room temperature at low speed (1,500 g) for 5 min and resuspended in BMMY medium to an OD 600 of 1.0.
  • a total volume of 1/200 of 100% methanol (final concentration 0.5%) was added to the medium and incubated at 28 ° C, 250 rpm for 72 hr, with a total volume of 1/200 of 100% methanol added per 24 hr.
  • the centrifuge was centrifuged at a low speed (1,500 g) and the supernatant was collected.
  • SDS-PAGE (Invitrogen, Cat. No. 456-1083) was used to detect protein expression by electrophoresis, and a better clone was selected for further fermentation.
  • the resulting recombinant human relaxin 800800 sequence (WT, wild type) is as follows:
  • Relaxin 800801 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • PCR site-directed mutagenesis using the relaxin 800800 vector as a template using KOD plus kit (TOYOBO, Cat.KOD-201) 25 ⁇ L reaction system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, primers 1, 2 (10 ⁇ M) 1 ⁇ L, 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4, 16.5 ⁇ L ddH 2 O.
  • the reaction procedure was 94 ° C for 5 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 12 minutes, amplification for 30 cycles, and then 68 ° C for 12 minutes to end the PCR amplification procedure.
  • DH5 ⁇ competent cells Teangen Biochemistry, Cat. CB101-02 were transformed, and positive clones were selected for sequencing analysis by Invitrogen.
  • the 801 precursor DNA sequence is as follows:
  • the resulting recombinant human relaxin 800801 sequence (WT, wild type) is as follows:
  • Relaxin 800802 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • PCR site-directed mutagenesis using the relaxin 800801 vector as a template using KOD plus kit (TOYOBO, Cat.KOD-201) 25 ⁇ L reaction system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, primers 1, 2 (10 ⁇ M) each 1 ⁇ L 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4, 16.5 ⁇ L ddH 2 O.
  • the reaction procedure was 94 ° C for 5 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 12 minutes, amplification for 30 cycles, and then 68 ° C for 12 minutes to end the PCR amplification procedure.
  • DH5 ⁇ competent cells Teangen Biochemistry, Cat. CB101-02 were transformed, and positive clones were selected for sequencing analysis by Invitrogen.
  • the 802 precursor DNA sequence is as follows:
  • the resulting recombinant human relaxin 800802 sequence (WT, wild type) is as follows:
  • Relaxation 800802-1 was performed by site-directed mutagenesis by PCR and synthesized by Invitrogen 4 A single-stranded DNA fragment is used as a site-directed mutagenesis primer with the following sequence:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800802 vector as a template, and the mutation process was the same as in Example 2.
  • the 800802-1 precursor DNA sequence was as follows:
  • Relaxin 800803 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800801 vector as a template, and the mutation process was the same as in Example 2.
  • the 800803 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin 800803 sequence (WT, wild type) is as follows:
  • the relaxin 800803-1 was subjected to site-directed mutagenesis by PCR, and four single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800803 vector as a template, and the mutation process was the same as in Example 2.
  • the 800803-1 precursor DNA sequence was as follows:
  • the relaxin 800805 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800801 vector as a template, and the mutation process was the same as in Example 2.
  • the 800805 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin 800805 sequence (WT, wild type) is as follows:
  • the relaxin 800805-1 was subjected to site-directed mutagenesis by PCR, and four single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800805 vector as a template, and the mutation process was the same as in Example 2.
  • the 800805-1 precursor DNA sequence was as follows:
  • Relaxin 800806 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800801 vector as a template, and the mutation process was the same as in Example 2.
  • the 800806 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin 800806 sequence (WT, wild type) is as follows:
  • the relaxin 800806-1 was subjected to site-directed mutagenesis by PCR, and four single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800806 vector as a template, and the mutation process was the same as in Example 2.
  • the 800806-1 precursor DNA sequence was as follows:
  • Relaxin 800808 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800800 vector as a template, and the mutation process was the same as in Example 2.
  • the 800808 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin 800808 sequence (WT, wild type) is as follows:
  • Example 12 Molecular cloning and expression of recombinant human relaxin 800808-1
  • the relaxin 800808-1 was subjected to site-directed mutagenesis by PCR, and four single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800808 vector as a template, and the mutation process was the same as in Example 2.
  • the 800808-1 precursor DNA sequence was as follows:
  • Relaxin 800809 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800800 vector as a template, and the mutation process was the same as in Example 2.
  • the 800809 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin 800809 sequence (WT, wild type) is as follows:
  • the relaxin 800809-1 was subjected to site-directed mutagenesis by PCR, and four single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers with the sequence relaxin 800808-1:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800809 vector as a template, and the mutation process was the same as in Example 2.
  • the 800809-1 precursor DNA sequence was as follows:
  • Relaxin 800810 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800800 vector as a template, and the mutation process was the same as in Example 2.
  • the 800810 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin analog 800810 sequence is as follows:
  • the relaxin 800810-1 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800810 vector as a template, and the mutation process was the same as in Example 2.
  • the 800810-1 precursor DNA sequence was as follows:
  • the resulting recombinant human relaxin analog 800810-1 sequence is as follows:
  • Relaxin 800811 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800804 vector as a template, and the mutation process was the same as in Example 2.
  • the 800811 precursor DNA sequence was as follows:
  • Relaxation 800813 was performed by site-directed mutagenesis by PCR and synthesized by Invitrogen 2 Single-stranded DNA fragments are used as site-directed mutagenesis primers with the following sequence:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800811 vector as a template, and the mutation process was the same as in Example 2.
  • the 800813 precursor DNA sequence was as follows:
  • Relaxin 800814 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800811 vector as a template, and the mutation process was the same as in Example 2.
  • the 800814 precursor DNA sequence was as follows:
  • the resulting test sample of the present disclosure has a recombinant human relaxin analog 800814 sequence as follows:
  • Relaxin 800816 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800804 vector as a template, and the mutation process was the same as in Example 2.
  • the 800816 precursor DNA sequence was as follows:
  • Example 21 Molecular cloning and expression of recombinant human relaxin 800847Y
  • Relaxation 800847Y was fixed by site-directed mutagenesis by PCR and synthesized by Invitrogen 2 A single-stranded DNA fragment is used as a site-directed mutagenesis primer with the following sequence:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800814 vector as a template, and the mutation process was the same as in Example 2.
  • the 800847Y precursor DNA sequence was as follows:
  • Example 22 Molecular cloning and expression of recombinant human relaxin 800851Y
  • the relaxin 800851Y was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800814 vector as a template, and the mutation process was the same as in Example 2.
  • the 800851Y precursor DNA sequence was as follows:
  • the codon-optimized human relaxin DNA sequence was introduced with a NdeI restriction site (CATATG) at the 5' end and a BamHI restriction site (GGATCC) at the 3' end.
  • the entire sequence was synthesized using the Overlap PCR method.
  • Six single-stranded DNA fragments were synthesized by Invitrogen as synthetic primers with the following sequence:
  • Relaxin synthesis was performed using a KOD plus kit (TOYOBO, Cat. KOD-201) in two-step PCR.
  • First step PCR 25 ⁇ L reaction system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, primers 1, 2, 3, 4, 5, 6 (10 ⁇ M) each 1 ⁇ L, 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4, 12.5 ⁇ L ddH 2 O .
  • the reaction procedure was 94 ° C for 5 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 30 seconds, amplification for 30 cycles, and then 68 ° C for 30 minutes to end the PCR amplification procedure.
  • the second step PCR 25 ⁇ L reaction system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, 1 ⁇ L of primers 1 and 6 (10 ⁇ M), 1 ⁇ L of the first round PCR product, 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4 , 15.5 ⁇ L ddH 2 O .
  • Reaction process The sequence was 94 ° C for 5 minutes, 94 ° C for 30 seconds, 68 ° C for 60 seconds, amplification for 30 cycles, and then 68 ° C for 10 minutes to end the PCR amplification procedure.
  • the synthesized DNA sequence and the pET9a (Novagen, Cat. 64431-3) vector were separately digested with NdeI/BamHI (Takara, Cat. D1161A/D1010A), and the obtained fragment was recovered on a 1.2% agarose gel using T4 DNA.
  • the ligase (New England Biolabs, Cat. M0202V) was ligated to transform DH5 ⁇ competent cells (Tiangen Biochemistry, Cat. CB101-02), and positive clones were selected for sequencing by Invitrogen.
  • the 800828 precursor DNA sequence is as follows:
  • the correctly sequenced pET9a-relaxin 800828 plasmid was transformed into BL21(DE3) competent cells (Tiangen Biochemistry, Cat. CB105-02), and the monoclonal strain was picked for IPTG-induced expression.
  • the specific method is as follows: a monoclonal strain is picked from a fresh plate, and added to 10 ml of LB medium containing ampicillin, and shake cultured at 37 ° C until the OD600 value reaches 0.6. A portion of the sample was taken out as an uninducible control and the cells were cryopreserved. A 1 M IPTG concentrated stock solution was added to the remaining sample to a final concentration of 1 mM, and incubation was continued for 4 hours. After the end of induction, the cells were collected by centrifugation and analyzed by SDS-PAGE electrophoresis.
  • the resulting recombinant human relaxin 800828 sequence (WT, wild type) is as follows:
  • the relaxin 800843 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800828 vector as a template, and the mutation process was the same as in Example 18. 800843
  • the precursor DNA sequence is as follows:
  • Example 25 Molecular cloning and expression of recombinant human relaxin 800847
  • Relaxin 800847 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the PCR site-directed mutagenesis was carried out using the relaxin 800845 vector as a template, and the mutation process was the same as in Example 18.
  • the 800847 precursor DNA sequence was as follows:
  • the relaxin 800851 was subjected to site-directed mutagenesis by PCR, and two single-stranded DNA fragments were synthesized by Invitrogen as site-directed mutagenesis primers.
  • the sequences are as follows:
  • the site-directed mutagenesis of PCR was carried out using the relaxin 800845 vector as a template, and the mutation process was the same as in Example 18.
  • the 800851 precursor DNA sequence was as follows:
  • LB medium Yeast extract 5g / L, yptone 10g / L, NaCl 5g / L, 121 ° C 30min high temperature sterilization,
  • Seed activation The seed glycerol tube stored at -80 ° C was thawed at room temperature, 500 ⁇ L of glycerol tuberculosis solution was aseptically added to 50 mL of LB medium, and 5 ⁇ L of kanamycin mother liquor was added thereto, and cultured at 37 ° C and 200 rpm for 7 hours.
  • the OD600 of the fermentation broth is sampled at intervals of about 1 hour until the OD 600 of the fermentation broth is ⁇ 40, and induction is started.
  • IPTG (1mol/L, sterile filtration through 0.22 ⁇ m filter before use), add 4mL at a time to the final concentration of IPTG 1mmol / L, the temperature of the induction phase is controlled at 37 ° C, other conditions remain unchanged, Induction for 12 hours.
  • the strain After a period of adaptation (10-12hr), the strain enters the exponential growth phase. By continuously increasing the stirring speed and aeration amount to meet the DO>30% required for the growth of the cells, the rotation speed is increased by 50-100 rpm each time. Controlled fermentation temperature: 37 ° C, tank pressure: 0.04-0.05 MPa, pH: 7.0.
  • Escherichia coli wet weight 100 g was suspended in 300 ml of 50 mM Tris pH 8.5, 0.2% EDTA, and ultrasonically disrupted. Centrifuge at 10,000 rpm for 4 hr at 4 ° C and discard the supernatant.
  • RP-UPLC conditions Waters UPLC BioHClass, ACQUITY UPLC BEH 300C4 (1.7 ⁇ m 2.1 ⁇ 100 mm), mobile phase: A solution 0.1% TFA; B solution ACN, flow rate 0.3 ml / min, linear gradient: 0.5 min (10B %) to 9min (60B%).
  • IEC-HPLC Waters UPLC BioHClass, Protein-Pak Hi Res CM (7 ⁇ m, 4.6 ⁇ 100 mm), Liquid A: 20 mM HEPES pH 8.0; Liquid B 0.5 M NaCl 20 mM HEPES pH 8.0, linear gradient 1 min (0%) B) to 10 min (100% B).
  • Sample 800828 (WT) of the original sequence was dissolved at 20 mM NaAc pH 5.0 for activity assay.
  • the resulting recombinant human relaxin 800828 sequence (WT, wild type) is as follows:
  • Step 1 the supernatant is purified by column
  • the fermentation broth supernatant was first purified on a Capto MMC (GE17-5318-03) column using an AKTA purifier.
  • the column was equilibrated with 20 mM NaAC pH 4, and the supernatant of the fermentation broth was adjusted to pH 4 and loaded.
  • the elution peak was collected by eluting with 100 mM NaHCO 3 pH 11.
  • purification was continued using RP-HPLC.
  • the desired product was obtained by lyophilization, and the purity by HPLC was 93%.
  • the molecular weight of the product was determined by LC-MS to be 5953.0, which was consistent with the calculated molecular weight of 5952.9.
  • the sequence of the recombinant human relaxin analog 800814 was as follows:
  • Phase A water (containing 0.1% formic acid); phase B: acetonitrile (containing 0.1% formic acid),
  • Mass spectrometry conditions were: ion source: AJS ESI (+), ion source parameters: Nebulizer 40 psig, Drying Gas Temp 325 ° C, Gas Flow 10 l/min, Sheath Gas Temp 350 ° C, Sheath Gas Flow 12 l/min, Vcap 3500 V, Fragmentor 200V , scanning range: m / z 50-3200.
  • Molecular weight determination of relaxin 800814 The molecular weight was determined to be 5948.7996 Da, which was completely consistent with the theoretical value, and the protein expression was confirmed to be correct.
  • Relaxin peptide map analysis of relaxin 800814 Sequence coverage analysis was performed using DTT reducing protein disulfide bond to detect whether protein expression was correct. The conditions were as follows: A 0.1 mg/mL sample was added to a final concentration of 20 mM DTT for 2 h, and then added to a final concentration of 40 mM IAA for 30 min. The treated samples were subjected to LC-MS detection and analysis. The results showed that the molecular sequences were perfectly matched, further demonstrating that the expressed protein molecules were as expected.
  • recombinant human relaxin 800814 (SEQ ID NO: 19, prepared in Example 19) in a 50 mL reaction tube, and add 5.0 mL of 0.1 M CH 3 COOH at pH 4.5 and 4 ° C. / CH 3 COONa buffer solution samples, recombinant human relaxin 800,814 final concentration of 2.0mg / mL (0.1M acetic acid / sodium acetate formulation: 3.7g sodium acetate in glacial acetic acid + 3.2g water + 1000mL).
  • the purifier 10 high performance liquid chromatography system was separated and purified from the recombinant human relaxin 800814-PEG derivative (referred to as PEG-814) by SP Sepharose Fast Flow cation exchange medium chromatography column (1.6 cm * 18 cm).
  • the equilibration buffer solution was HAC-NaAC with a pH of 4.5 and a concentration of 20 mmol/L. After 10 times dilution, the sample was collected for peak flow, and then eluted with a buffer containing 1 mol/L of NaCl to collect an elution peak.
  • the flow rate was 1.0 mL/min, and the detection wavelength was 280 nm.
  • the human relaxin analog derivative PEG-814 of the present disclosure was obtained.
  • Test Example 1 Detection of human biological activity in vitro of human relaxin and its analogues
  • the human relaxin analog (800814) of the present disclosure binds to a receptor on THP-1 cells, inducing THP-1 cells to produce cAMP.
  • the in vitro activity of the human relaxin analog (800814) was determined by measuring the amount of cAMP production.
  • the original sequence of wild-type human relaxin analog 800828 (WT) (prepared in Example 29) was used in this test as a positive control.
  • THP-1 (ATCC, Product Number: TIB-202 TM) at 37 °C, 5% CO 2 grow in suspension, when the cells are in logarithmic growth phase state is better, or may be used for passaging experiments. It is passed every 2 to 3 days, with a ratio of 1:3 to 1:4. Medium: RPMI 1640 + 0.05 mM b-mercaptoethanol + 10% FBS. On the day of the experiment, the THP-1 cells in the logarithmic growth phase were collected by centrifugation, resuspended in DMEM/F12, and adjusted to a cell density of 2 ⁇ 10 6 cells/ml in 96-well plates (50 ⁇ l per well), 37 ° C incubator.
  • the human relaxin analog was diluted in a 3-fold ratio with a diluent (DEME/F12 + 0.2% BSA + 0.02 polysorbate 80 + 2 ⁇ M forskolin + 500 ⁇ M IBMX). Add 50 ⁇ l of the diluted drug to the 96-well plate, mix for 2 minutes, and incubate for 30 minutes in a 37 ° C incubator.
  • a diluent DEME/F12 + 0.2% BSA + 0.02 polysorbate 80 + 2 ⁇ M forskolin + 500 ⁇ M IBMX.
  • Table 5 In vitro activity assay of human wild-type relaxin (800828) and human relaxin analog (800814)
  • the 800814 precursor molecule can not only be digested in the cell (eliminating the in vitro digestion step of the expression product), but also the mature relaxin analog directly secreted into the supernatant (800814).
  • the in vitro cell viability was doubled compared to the positive molecule (800828 (WT)) (EC50 increased from 3.05 ng/ml to 1.50 ng/ml).
  • Test Example 2 Detection of biological activity of human relaxin and its analogues in vivo
  • mice female, purchased from Sipple Bikai Experimental Animal Co., Ltd., license: SCXK (Shanghai) 2008-0016, 18-20 g); feeding environment: SPF grade. After the mice were purchased, the laboratory environment was kept for 2 weeks, with 12/12 hour light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%.
  • the original sequence of wild-type human relaxin analog 800828 was used in this test as a positive control.
  • each ICR female was injected subcutaneously with 5 ⁇ g/100 ⁇ l/ ⁇ -estradiol (water insoluble, slightly soluble in oil) and mixed with olive oil. After 6 days, ICR mice weighing less than 22 g were removed and the remainder were grouped by weight.
  • Relaxin 800828 (WT) (6 ⁇ g/100 ⁇ l/mouse), 800814 (6 ⁇ g/100 ⁇ l/mouse) or 0.1% benzo red Violet 4B physiological saline (solvent control group) was injected subcutaneously. Relaxin 800828 (WT) and 800814 were both dissolved in physiological saline containing 0.1% benzopurpurin 4B.
  • the pubis was taken, excess skin and muscle were removed, and the width of the pubic bone was measured under a microscope; the mean value of the pubic bone width in the solvent control group was 1, and the ratio of each administration group was the relative width.
  • Test Example 3 Determination of half-life in vivo of human relaxin and its analogues
  • the experimental SD rats male and female, a total of 12, were purchased from Xipuer Bikai Experimental Animal Co., Ltd., license: SCXK (Shanghai) 2008-0016, 160-180g, feeding environment: SPF level. SD rats were taken for 3 days in a laboratory environment at a temperature of 20-25 ° C and a humidity of 40-60%.
  • the original sequence of wild-type human relaxin analog 800828 was used in this test as a positive control.
  • Rats were randomly divided into 3 groups, 4 in each group, and the number of males and females was the same.
  • the control serum
  • human relaxin analog 800814, WT 8008248
  • the test samples were taken from the eyelids 0, 0.03, 0.08, 0.25, 0.5, 1, 1.5, 2, 4, 6, and 8 hours after the injection of the rats.
  • the blood sample was centrifuged, and the supernatant was taken and stored at -20 ° C for testing. After the blood samples were collected, the content of relaxin was measured using a human relaxin-2 Quantikine ELISA Kit (R&D).
  • the T 1/2 of the drug to be tested is calculated using the T1/2 calculation formula and EXCEL. The results are as follows:
  • Test Example 4 Detection of long-acting activity of human relaxin analog derivative PEG-814 in vivo
  • the rats were purchased, they were housed in 5 cages, housed in a laboratory environment for 1 week, adjusted for 12/12 hours of light/dark cycle, temperature 20-25 ° C; humidity 50-60%.
  • the basal blood pressure of SHR rats was tested 2-3 times with a non-invasive sphygmomanometer (provided by Softron, model BP-98A), and rats with stable blood pressure and not less than 170 mmHg were randomly divided into test group according to blood pressure. PEG-814) and solvent control group (physiological saline), 10 in each group.
  • the rats were intravenously injected with relaxin PEG-814 or physiological saline, 500 ⁇ l each (30 ⁇ g/day/rat), once daily (15:00 p.m.) for 6 weeks. Blood pressure was measured weekly and body weight and blood pressure data were recorded.
  • the changes of body weight and blood pressure were measured by excel software.
  • the body weight and blood pressure data of the test group and the solvent group were tested by t test.
  • the blood pressure of the drug-administered group and the control group was statistically significant. To evaluate the blood pressure lowering effect of PEG-814.

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Abstract

本发明公开了人松弛素类似物、编码所述人松弛素类似物的多核苷酸、其药物组合物以及其在医药上的应用，还公开了所述人松弛素类似物的衍生物。

Description

人松驰素类似物、其药物组合物及其在医药上的应用 技术领域
本公开涉及一种新型人松驰素类似物，包含其的药物组合物及其用途。
背景技术
松弛素(Relaxin简称RLX)是一种多肽激素，其由哺乳动物的卵巢黄体分泌。松弛素在体内具有多种生理功能，包括松弛耻骨韧带、抑制子宫收缩、软化子宫颈、刺激乳腺发育、影响乳汁分泌。1926年，在研究妊娠期间骨盆带变化时，松弛素由Frederick Hisaw首次发现。20世纪70至80年代，松弛素被认为是一种双链蛋白质。松弛素的结构与胰岛素相似。已经证实松弛素是肽类激素家族的成员之一。在人类当中，松弛素家族由7个基因所编码：RLN1、RLN2、RLN3(也作INSL7)、INSL3/RLF、INSL4/EPIL、INSL5/RIF2和INSL6/RIF1。人体循环中的大多数人类松弛素是基因RLN2编码的产物。RLN2的翻译产物是松弛素前体，该松弛素前体包括(从N端至C端)：24个氨基酸残基的信号肽序列、29个氨基酸残基的B链、104-107个氨基酸残基的连接序列，以及约24个氨基酸残基的A链。
目前已明确，松弛素不仅在妊娠过程中发挥重要作用，而且对非妊娠动物的血管结构和功能也产生影响。松弛素具有广泛的生物学作用，包括在哺乳动物妊娠和机体老化过程中维持内环境的稳定、抗炎、心肌保护、扩张血管、促进伤口愈合，尤为重要的作用是抗纤维化。各种原因引起的心脏、肾脏病变最终均导致纤维化、结构改变、功能丧失的发生。因此，纤维化过程的有效抑制，是挽救脏器功能的重要措施。鉴于此，松弛素的抗纤维化作用可能成为未来抗纤维化治疗的有效措施。更为重要的是，松弛素可由心脏产生，并通过局部受体来保护心脏和调节细胞外基质。松弛素已成功地用于改善多种动物模型的心脏纤维化。因而，松弛素有望用于人类心脏纤维化性疾病的治疗(杜晓军，周娟，Baker心脏研究所)。
现有技术中，在大肠杆菌中表达人松弛素(hRelaxin,野生型)前体；经过纯化后前体折叠复性；两步酶切(分别用CPB和胰蛋白酶)；再纯化得到有活性的产物松弛素。现有技术中的缺陷是：生产过程中需要蛋白复性、两步酶切和再纯化。繁多的步骤导致产量大大降低以及成本的提高，因为每一步都会涉及蛋白的损失。而且，外源酶(CPB、胰蛋白酶等)本身的成本高，酶的质量也会影响终产品的质量和产量。
另一缺陷是，所表达的野生型松弛素活性低。这使得治疗剂量所需的重组蛋白的量增多，成本增加；而且所表达的野生型松弛素的低活性，给进一步开发蛋白修饰产品带来困难，因为修饰会损失蛋白的活性。
针对现有技术的上述缺陷，通过对野生型松弛素的特定位点氨基酸改变，本公开进行了新型分子的设计，并发现了新型的人松弛素类似物，从而能非常好的 解决上述问题。新的技术方案显示以下优点：
1.松弛素特定位点的氨基酸改变，使所得的类似物能够在酵母细胞内完成正确的折叠，在这一过程中细胞自身的酶系统除去A和B链中的c-肽段。得到的完整有功能的活性分子被分泌到细胞外。通过一步纯化得到活性分子。省去现有技术所需的表达后复性、酶切、纯化等多个步骤。
2.松弛素特定位点的氨基酸改变，使结构更加适应细胞内折叠、酶切和分泌，而且提高了生物学活性。这样可以减少单位剂量所需的重组蛋白质的量，从而降低成本。同时，因为活性提高，使得这样的类似物更加容易分子修饰物的开发。
通过设计(松弛素序列中特定氨基酸的替换、缺失等)，本公开得到特定序列的人松驰素类似物。这种类似物能在真核表达系统(细胞)里完成松弛素前体的蛋白折叠、酶切；并且分泌到发酵液中时，本公开的人松驰素类似物是成熟的、完整、有功能的松弛素类似物。不仅如此，通过这些特定的设计所得到的松弛素类似物，其生物学活性比野生型松弛素高2倍以上。
发明内容
本公开提供一种人松弛素类似物，其包含A链和B链，所述A链和B链的氨基酸序列分别如下式所示：
A链：A1LYSALANKCCHVGCTKRSLARFC(24个氨基酸残基)
B链：DSWMEEVIKLCGRB14LVRAQIAICGMSTWS(29个氨基酸残基)
其中A1选自：Q、D、E和W；
其中B14选自：E、D和N；
任选地，B1和B2同时缺失；当A1为Q时，B14不为E或D。
在本公开的一个实施方案中，提供一种如上所述的人松弛素类似物，其包含A链和B链，所述A链和B链的氨基酸序列分别如下式所示：
A链：A1LYSALANKCCHVGCTKRSLARFC(24个氨基酸残基)
B链：DSWMEEVIKLCGRB14LVRAQIAICGMSTWS(29个氨基酸残基)
其中A1选自：Q、D、E和W；
其中B14选自：E、D和N；
当A1为Q时，B14不为E或D。
在本公开一个实施方案中，所述的A1为D、E或W，优选为D。
在本公开一个实施方案中，所述的A1为D，B14为D。具体地，实例为SEQ IDNO:134(A链)和SEQ ID NO:135(B链)，通过二硫键连接而形成的人松弛素类似物。
在本公开一个实施方案中，所述的B14为D。
在本公开一个实施方案中，一种如上所述的人松弛素类似物，其中所述的A链和B链的氨基酸序列选自如下组合：
Figure PCTCN2014088280-appb-000001
在本公开一个实施方案中，一种如上所述的人松弛素类似物，其中所述B链通过连接序列(本公开中简称L)与所述A链连接，所述连接序列的长度为1至15个氨基酸残基，优选为2至8个氨基酸残基，所述的连接序列的氨基酸序列选自：
Figure PCTCN2014088280-appb-000002
在本公开一个实施方案中，一种如上所述的人松弛素类似物，其中所述的人松弛素类似物的N端连接有信号肽序列(本公开中简称S)，所述信号肽序列的长度为4至15个氨基酸残基，优选6至11个氨基酸残基；更优选的，所述的信号肽序列的氨基酸序列选自：
S1：EEGEPK，          SEQ ID NO:33，
S2：EEGEPKR，         SEQ ID NO:34，和
S3：MKKNIAFLLKR    SEQ ID NO:35。
在本公开一个实施方案中，一种用于制备如上所述的人松弛素类似物的表达前体，表达前体的氨基酸序列选自SEQ ID NO:1至SEQ ID NO:26的一种或多种。
表1.人松弛素类似物表达前体汇总
Figure PCTCN2014088280-appb-000003
Figure PCTCN2014088280-appb-000004
Figure PCTCN2014088280-appb-000005
本公开进一步提供一种人松弛素类似物的衍生物，其是如上所述的人松弛素类似物经PEG化修饰所得，换言之其中如上所述的人松弛素类似物被PEG分子所修饰。在一些实施方案中，所述的PEG分子的分子量为5-100KDa；在一些实施方案中，是10-80KDa；在一些实施方案中，15-45KDa；在一些实施方案中，20-40KDa；在一些实施方案中，所述的PEG分子为支链型或直链型。
本公开进一步提供一种多核苷酸，其编码如上所述的人松弛素类似物的表达前体。多核苷酸选自DNA或RNA。技术人员理解，多核苷酸的互补序列也涵盖在本公开的范围内。
本公开进一步提供一种含有如上所述多核苷酸的表达载体。
本公开进一步提供一种转化有如上所述的表达载体的宿主细胞。在一些实施方案中，所述的宿主细胞为细菌，在一些具体实施方案中为大肠杆菌；在另一些实施方案中，宿主细胞为酵母菌，在一些具体实施方案中为毕赤酵母。
本公开进一步提供一种药物组合物，其含有或由如下组成：
a)一种或多种如上所述的人松弛素类似物、和/或一种或多种如上所述的人松弛素类似物衍生物，和
b)一种或多种药学上可接受的载体、稀释剂或赋形剂。
本公开进一步提供一种人松弛素类似物或其衍生物的可注射溶液，其含有溶解形式或可溶解形式的如上所述的药物组合物。应当理解，可注射溶液的干粉或冻干粉形式也涵盖在本公开的范围内。
本公开进一步提供如上所述的人松弛素类似物、如上所述的人松弛素类似物衍生物、或如上所述的药物组合物、或如上所述的可注射溶液，在制备用于治疗或预防纤维化疾病或心血管疾病的药物中的用途，参见Cardiovascular effects of relaxin；from basic science to clinical therapy，Nat.Rev.Cardiol.7,48-58(2010)；Relaxin decreases renal interstitial fibrosis and slows progression of renal disease，Kindney International，Vol.59(2001),pp.876-882。
本公开进一步提供一种治疗或预防纤维化疾病或心血管疾病的方法，该方法包括向所需的受试者施用治疗有效量的如上所述的人松弛素类似物或其衍生物，或如上所述的药物组合物。
发明详述
一、术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，“给予”、“施用”、“治疗”和“处理”是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”、“治疗”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”、“治疗”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞，在体外和离体处理细胞。当应用于人、兽医学或研究受试者时，“处理”是指治疗处理、预防或预防性措施、研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如给予患者包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂。所述的有效缓解的量包括无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个患者都有的目标疾病症状方面，可能效果不同，但是根据本领域已知的任何统计学检验方法，如Student t检验、卡方检验、Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验，确定其在患者中应当统计学意义上显著减轻目标疾病症状。
除非另有说明，否则在本公开中所述的松弛素或人松驰素为人松驰素RLN2，其包含具有生物活性的全长序列或部分序列。人松驰素包含A链和B链，两者之间通过二硫键结合。本公开中所用人松驰素的序列描述参见GenBank序列号：EAW58770。本公开中用于作为阳性对照的人松驰素800828为野生型。
本公开中所述的人松驰素类似物前体按照从氨基端至羧基端的顺序包含：信号肽序列、B链或其突变体、连接序列、A链或其突变体。其中信号肽序列的长度是4-15个氨基酸残基，优选6-11个氨基酸残基；B链或其变体的长度是29个氨基酸残基；连接序列的长度是1-15个氨基酸残基，优选2-8个氨基酸残基；以及A链或其变体的长度是约24个氨基酸残基。
本公开中所述的突变体指在序列上进行氨基酸的修饰、替换、置换或取代。本公开的B链优选的突变为在B链第14位(简称B14)上将E替换为D，且A链第1位(简称A1)Q替换为D。
本公开的序列缩写中，Ln表示本公开中的某种连接序列(如L1至L6)，Sn表示本公开中的某种信号肽序列(如S1、S2、S3)；DA1表示在A链第1个氨基酸突变为D，DB14表示在B链第14个氨基酸突变为D，DelB1,B2表示B链第1和2个氨基酸缺失；A(wt)，表示野生型无突变的A链，B(wt)，表示野生型无突变的B链。
“保守修饰”或“保守置换或取代”是指：蛋白中的氨基酸残基被具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸残基所置换；即使蛋白中的这种氨基酸残基被频繁改变，也不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，第4版)。另外，结构或功能类似的氨基酸残基的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法、途径、和剂量、以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指来源在生物、细胞或人体外产生的物质。“内源性”来源在细胞、生物或人体内产生的物质。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，表述“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。以上表述还包括与最初转化细胞具有相同功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指，例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，从而能够设计寡核苷酸引物；这些引物的序列与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列、以及由总细胞RNA转录而来的cDNA、噬菌体或质粒序列等。一般，参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合 酶反应法的一个实例，但不是唯一的实例；所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生。
“药物组合物”表示含有一种或多种本文所述类似物或前体以及其他化学组分的混合物，其中其他组分例如生理学/可药用的载体和赋形剂。其他化学组分的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本公开中所述的用转化宿主细胞的步骤可用本领域技术人员熟知的常规技术进行。获得的转化体可以用常规方法培养，其表达本公开的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。从表达前体产物中释放所述人松弛素类似物的化学和/或酶方法可用本领域技术人员熟知的常规技术进行，比如胰蛋白酶、羧肽酶B、赖氨酸内肽酶C等。
具体实施方式
实施例1、重组人松弛素800800的分子克隆和表达
1、重组人松弛素800800表达载体的构建
经过密码子优化的人松弛素800800的DNA序列通过Overlap PCR方法合成。由Invitrogen公司合成6条单链DNA片段作为合成引物，序列如下：
800-引物1
CTCGAGGATTCTTGGATGGAAGAAGTTATTAAGT          SEQ ID NO: 36
800-引物2
CAATTTGAGCTCTAACCAATTCTCTACCACACAACTTAATAACTTCTTCCATCCAAGAA                                        SEQ ID NO: 37
800-引物3
AGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGACA                                        SEQ ID NO: 38
800-引物4
ATGACAACACTTGTTAGCCAAAGCAGAGTACAATTGTCTCTTAGACCAAGTAGACATAC                                        SEQ ID NO: 39
800-引物5
CTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGT                                        SEQ ID NO: 40
800-引物6     GAATTCTTAACAAAATCTAGCCAAAGATCTCTTA
                                              SEQ ID NO: 41
松弛素的合成采用KOD plus试剂盒(TOYOBO, Cat. KOD-201)，采用两步PCR完成。
第一步PCR，25μL反应体系：2.5μL 10×KOD 缓冲液，2.5μL 2mM dNTPs， 引物1,2,3,4,5,6(10μM)各1μL，0.5μL KOD plus，1μL 25mM MgSO4，12.5μLddH2O。
反应程序为94℃5分钟；94℃30秒钟，60℃30秒钟，68℃30秒钟，扩增30个循环后；再68℃30分钟以结束PCR扩增程序。
第二步PCR，25μL反应体系：2.5μL 10×KOD buffer，2.5μL 2mM dNTPs，引物1和6(10μM)各1μL，1μL第一轮PCR产物，0.5μL KOD plus，1μL 25mM MgSO4，15.5μL ddH2O。
反应程序为94℃5分钟；94℃30秒钟，68℃60秒钟，扩增30个循环后；再68℃10分钟以结束PCR扩增程序。
将PCR合成的DNA序列和pPIC9K表达载体(Invitrogen,Cat.K1750-01)分别进行EcoR I/Xho I(New England Biolabs,Cat.R0101S/R0146V)双酶切，得到的目的片段经1.2％琼脂糖凝胶回收，用T4DNA连接酶(New England Biolabs,Cat.M0202V)连接，转化DH5α感受态细胞(天根生化，Cat.CB101-02)，挑选阳性克隆由Invitrogen公司作测序分析。800前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA                               SEQ ID NO:42
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRQLYSALANKCCHVGCTKRSLARFC                             SEQ ID NO:1。
2、重组人松弛素800800表达载体的转化
将5-10μg的上述步骤中得到的松弛素800800表达载体用SalI(Takata,Cat.D1080a)进行线性化。再加入1/10体积的3M醋酸钠，2倍体积的无水乙醇，充分混合后，放置于-20℃，2hr。混合物高速离心(13,000rpm)5min，去处上清，用75％乙醇清洗沉淀2次，将沉淀倒置晾干后，用10μL ddH2O溶解。将线性化的质粒与80μL电转化感受态细胞(毕赤酵母GS115，Invitrogen,Cat.K1750-01)混合转入电转杯(Bio Rad,Cat.1652086)中，并置冰浴5min。将电转仪(Bio Rad Micropulser)参数设至2kV，25Ω，200uF，进行电转化。结束后迅速加入1ml冰浴的D-山梨醇(生工生物工程有限公司)混合，将100-300μl混合物涂布于MD培养板上，30℃培养3d至形成菌落。
3、重组人松弛素800800表达克隆的G418筛选
用3ml YPD培养基洗脱MD培养板上的菌落，重悬，并用分光光度计(Beckman,DU800)测定重悬的细胞浓度(1OD600＝5×107cell/ml)。将1×105细胞涂在4mg/ml浓度G418(GIBCO,Cat.11811-031)的YPD培养板上，30℃培养5d至形成菌落。
4、重组人松弛素800800的诱导表达
从YPD培养板上挑取单菌落置于4mL BMGY培养基，30℃，250rpm培养过 夜。第二天检测OD600值，应处于2-6之间。离心机(Beckman Coulter)室温低速(1,500g)离心5min收集细胞，并用BMMY培养基重悬至OD600为1.0。在培养基中加入1/200总体积的100％甲醇(终浓度为0.5％)，并于28℃，250rpm培养72hr，其中每24hr补加1/200总体积的100％甲醇。诱导结束后，离心机低速(1,500g)离心并收集上清。SDS-PAGE(Invitrogen,Cat.No.456－1083)电泳检测蛋白表达，挑选表达较好克隆进行下一步发酵。
得到的重组人松弛素800800序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS   SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例2、重组人松弛素800801的分子克隆和表达
1、重组人松弛素800801表达载体的构建
松弛素800801用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
801-引物1
GTATGTCTACTTGGTCTAAGAGATCTTTGAAGAGACAATTGTACTC
                                              SEQ ID NO:43
801-引物2
GAGTACAATTGTCTCTTCAAAGATCTCTTAGACCAAGTAGACATAC
                                              SEQ ID NO:44
PCR定点突变以松弛素800800载体为模板，采用KOD plus试剂盒(TOYOBO,Cat.KOD-201)25μL反应体系：2.5μL 10×KOD缓冲液，2.5μL 2mM dNTPs，引物1,2(10μM)各1μL，0.5μL KOD plus，1μL 25mM MgSO4，16.5μL ddH2O。反应程序为94℃5分钟，94℃30秒钟，60℃30秒钟，68℃12分钟，扩增30个循环后，再68℃12分钟以结束PCR扩增程序。在PCR产物中直接加入内切酶DpnI(NEB Cat.R0176L)1μL消化5小时后，转化DH5α感受态细胞(天根生化，Cat.CB101-02)，挑选阳性克隆由Invitrogen公司作测序分析。801前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGATCTTTGAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA                 SEQ ID NO:45
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRSLKRQLYSALANKCCHVGCTKRSLARFC                                   SEQ ID NO:2
2、重组人松弛素800801的转化、筛选、诱导表达
重组人松弛素800801的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800801序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS   SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:135。
实施例3、重组人松弛素800802的分子克隆和表达
1、重组人松弛素800802表达载体的构建
松弛素800802用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
802-引物1
GTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAAGAGACAATTGTACTCTGC                          SEQ ID NO:46
802-引物2
GCAGAGTACAATTGTCTCTTCTTTCTAGAACCGTAACCAGTTGGCTTTCTCTTAGACCAAGTAGAC                              SEQ ID NO:47
PCR定点突变以松弛素800801载体为模板，采用KOD plus试剂盒(TOYOBO,Cat.KOD-201)25μL反应体系：2.5μL 10×KOD buffer，2.5μL 2mM dNTPs，引物1,2(10μM)各1μL，0.5μL KOD plus，1μL 25mM MgSO4，16.5μL ddH2O。反应程序为94℃5分钟，94℃30秒钟，60℃30秒钟，68℃12分钟，扩增30个循环后，再68℃12分钟以结束PCR扩增程序。在PCR产物中直接加入内切酶DpnI(NEB Cat.R0176L)1μL消化5小时后，转化DH5α感受态细胞(天根生化，Cat.CB101-02)，挑选阳性克隆由Invitrogen公司作测序分析。802前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
                                              SEQ ID NO:48
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSRKKRQLYSALANKCCHVGCTKRSLARFC                                 SEQ ID NO:3。
2、重组人松弛素800802的转化、筛选、诱导表达
重组人松弛素800802的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800802序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例4、重组人松弛素800802-1的分子克隆和表达
1、重组人松弛素800802-1表达载体的构建
松弛素800802-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4 条单链DNA片段作为定点突变引物，序列如下：
802-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
                                         SEQ ID NO:49
802-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                         SEQ ID NO:50
802-1-引物3GTTCTAGAAAGAAGAGAGATTTGTACTCTGCTTTGG
                                         SEQ ID NO:51
802-1-引物4CCAAAGCAGAGTACAAATCTCTCTTCTTTCTAGAAC
                                         SEQ ID NO:52
PCR定点突变以松弛素800802载体为模板，突变过程同实施例2。800802-1前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
                                         SEQ ID NO:53
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSRKKRDLYSALANKCCHVGCTKRSLARFC                        SEQ ID NO:4。
2、重组人松弛素800802-1的转化、筛选、诱导表达
重组人松弛素800802-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800802-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)   DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例5、重组人松弛素800803的分子克隆和表达
1、重组人松弛素800803表达载体的构建
松弛素800803用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
803-引物1
GTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAGACAATTGTACTCTGC            SEQ ID NO:54
803-引物2
GCAGAGTACAATTGTCTCTTTCTAGAACCGTAACCAGTTGGCTTTCTCTTAGACCAAGTAGAC            SEQ ID NO:55
PCR定点突变以松弛素800801载体为模板，突变过程同实施例2。800803前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA    SEQ ID NO:56
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSRKRQLYSALANKCCHVGCTKRSLARFC                           SEQ ID NO:5。
2、重组人松弛素800803的转化、筛选、诱导表达
重组人松弛素800803的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800803序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS   SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:135。
实施例6、重组人松弛素800803-1的分子克隆和表达
1、重组人松弛素800803-1表达载体的构建
松弛素800803-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4条单链DNA片段作为定点突变引物，序列如下：
803-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
SEQ ID NO:57
803-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                        SEQ ID NO:58
803-1-引物3CGGTTCTAGAAAGAGAGATTTGTACTCTGCTTTGG
                                        SEQ ID NO:59
803-1-引物4CCAAAGCAGAGTACAAATCTCTCTTTCTAGAACCG
                                        SEQ ID NO:60
PCR定点突变以松弛素800803载体为模板，突变过程同实施例2。800803-1前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAGAAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA。
SEQ ID NO:61
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSRKRDLYSALANKCCHVGCTKRSLARFC                     SEQ ID NO:6。
2、重组人松弛素800803-1的转化、筛选、诱导表达
重组人松弛素800803-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800803-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)   DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例7、重组人松弛素800805的分子克隆和表达
1、重组人松弛素800805表达载体的构建
松弛素800805用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
805-引物1
GTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAGAAGACAATTGTACTCTGCTTTG                                  SEQ ID NO:62
805-引物2
CAAAGCAGAGTACAATTGTCTTCTTGGACCACCACCTCTCTTAGACCAAGTAGAC                                   SEQ ID NO:63
PCR定点突变以松弛素800801载体为模板，突变过程同实施例2。800805前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAGAAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA           SEQ ID NO:64
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRGGGPRRQLYSALANKCCHVGCTKRSLARFC                                SEQ ID NO:7
2、重组人松弛素800805的转化、筛选、诱导表达
重组人松弛素800805的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800805序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例8、重组人松弛素800805-1的分子克隆和表达
1、重组人松弛素800805-1表达载体的构建
松弛素800805-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4条单链DNA片段作为定点突变引物，序列如下：
805-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
                                           SEQ ID NO:65
805-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                           SEQ ID NO:66
805-1-引物3GGTGGTCCAAGAAGAGATTTGTACTCTGCTTTGG
                                           SEQ ID NO:67
805-1-引物4CCAAAGCAGAGTACAAATCTCTTCTTGGACCACC
                                            SEQ ID NO:68
PCR定点突变以松弛素800805载体为模板，突变过程同实施例2。800805-1前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAGAAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA         SEQ ID NO:69
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRGGGPRRDLYSALANKCCHVGCTKRSLARFC                                  SEQ ID NO:8。
2、重组人松弛素800805-1的转化、筛选、诱导表达
重组人松弛素800805-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800805-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)   DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例9、重组人松弛素800806的分子克隆和表达
1、重组人松弛素800806表达载体的构建
松弛素800806用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
806-引物1
GTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAAGAGACAATTGTACTCTGCT
                                           SEQ ID NO:70
806-引物2
AGCAGAGTACAATTGTCTCTTTGGACCACCACCTCTCTTAGACCAAGTAGAC
                                           SEQ ID NO:71
PCR定点突变以松弛素800801载体为模板，突变过程同实施例2。800806前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA       SEQ ID NO:72
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRGGGPKRQLYSALANKCCHVGCTKRSLARFC                                SEQ ID NO:9。
2、重组人松弛素800806的转化、筛选、诱导表达
重组人松弛素800806的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800806序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:135。
实施例10、重组人松弛素800806-1的分子克隆和表达
1、重组人松弛素800806-1表达载体的构建
松弛素800806-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4条单链DNA片段作为定点突变引物，序列如下：
806-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
                                           SEQ ID NO:73
806-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                           SEQ ID NO:74
806-1-引物3GGTGGTGGTCCAAAGAGAGATTTGTACTCTGCTTTGG
                                           SEQ ID NO:75
806-1-引物4CCAAAGCAGAGTACAAATCTCTCTTTGGACCACCACC
                                        SEQ ID NO:76
PCR定点突变以松弛素800806载体为模板，突变过程同实施例2。800806-1前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGGTGGTGGTCCAAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA          SEQ ID NO:77
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRGGGPKRDLYSALANKCCHVGCTKRSLARFC                              SEQ ID NO:10
2、重组人松弛素800806-1的转化、筛选、诱导表达
重组人松弛素800806-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800806-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)  DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例11、重组人松弛素800808的分子克隆和表达
1、重组人松弛素800808表达载体的构建
松弛素800808用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
808-引物1
GGGTATCTCTCGAGAAAAGAGAAGAAGGTGAACCAAAGGATTCTTGGATGGAAGAAG                                  SEQ ID NO:78
808-引物2
CTTCTTCCATCCAAGAATCCTTTGGTTCACCTTCTTCTCTTTTCTCGAGAGATACCC                                       SEQ ID NO:79
PCR定点突变以松弛素800800载体为模板，突变过程同实施例2。800808前体DNA序列如下：
GAAGAAGGTGAACCAAAGGATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA     SEQ ID NO:80
上述DNA序列编码氨基酸序列如下：
EEGEPKDSWMEEVIKLCGRELVRAQIAICGMSTWSKRQLYSALANKCCHVGCTKRSLARFC                                 SEQ ID NO:11
2、重组人松弛素800808的转化、筛选、诱导表达
重组人松弛素800808的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800808序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:135。
实施例12、重组人松弛素800808-1的分子克隆和表达
1、重组人松弛素800808-1表达载体的构建
松弛素800808-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4条单链DNA片段作为定点突变引物，序列如下：
808-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
                                          SEQ ID NO:81
808-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                         SEQ ID NO:82
808-1-引物3CTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGG
                                         SEQ ID NO:83
808-1-引物4CCAAAGCAGAGTACAAATCTCTCTTAGACCAAGTAG
                                         SEQ ID NO:84
PCR定点突变以松弛素800808载体为模板，突变过程同实施例2。800808-1前体DNA序列如下：
GAAGAAGGTGAACCAAAGGATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA        SEQ ID NO:85
上述DNA序列编码氨基酸序列如下：
EEGEPKDSWMEEVIKLCGRDLVRAQIAICGMSTWSKRDLYSALANKCCHVGCTKRSLARFC                                           SEQ ID NO:12
2、重组人松弛素800808-1的转化、筛选、诱导表达
重组人松弛素800808-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800808-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS   SEQ ID NO: 137
A链(DA1) DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO: 138。
实施例13、重组人松弛素800809的分子克隆和表达
1、重组人松弛素800809表达载体的构建
松弛素800809用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
809-引物1
GGGTATCTCTCGAGAAAAGAGAAGAAGGTGAACCAAAGAGAGATTCTTGGATGGAAGAAG                                     SEQ ID NO:86
809-引物2
CTTCTTCCATCCAAGAATCTCTCTTTGGTTCACCTTCTTCTCTTTTCTCGAGAGATACCC                                     SEQ ID NO:87
PCR定点突变以松弛素800800载体为模板，突变过程同实施例2。800809前体DNA序列如下：
GAAGAAGGTGAACCAAAGAGAGATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA       SEQ ID NO:88
上述DNA序列编码氨基酸序列如下：
EEGEPKRDSWMEEVIKLCGRELVRAQIAICGMSTWSKRQLYSALANKCCHVGCTKRSLARFC                          SEQ ID NO:13。
2、重组人松弛素800809的转化、筛选、诱导表达
重组人松弛素800809的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素800809序列(WT，野生型)如下：
B链(WT) DSWMEEVIKLCGRELVRAQIAICGMSTWS   SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC       SEQ ID NO:135。
实施例14、重组人松弛素800809-1的分子克隆和表达
1、重组人松弛素800809-1表达载体的构建
松弛素800809-1用PCR的方法进行定点突变完成，由Invitrogen公司合成4条单链DNA片段作为定点突变引物，序列同松弛素800808-1：
809-1-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
                                      SEQ ID NO:89
809-1-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
                                     SEQ ID NO:90
809-1-引物3CTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGG
                                     SEQ ID NO:91
809-1-引物4CCAAAGCAGAGTACAAATCTCTCTTAGACCAAGTAG
                                     SEQ ID NO:92
PCR定点突变以松弛素800809载体为模板，突变过程同实施例2。800809-1前体DNA序列如下：
GAAGAAGGTGAACCAAAGAGAGATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA       SEQ ID NO:93
上述DNA序列编码氨基酸序列如下：
EEGEPKRDSWMEEVIKLCGRDLVRAQIAICGMSTWSKRDLYSALANKCCHVGCTKRSLARFC                          SEQ ID NO:14。
2、重组人松弛素800809-1的转化、筛选、诱导表达
重组人松弛素800809-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800809-1序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS   SEQ ID NO:137
A链(DA1) DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例15、重组人松弛素800810的分子克隆和表达
1、重组人松弛素800810表达载体的构建
松弛素800810用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
810-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
SEQ ID NO:94
810-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
SEQ ID NO:95
PCR定点突变以松弛素800800载体为模板，突变过程同实施例2。800810前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA         SEQ ID NO:96
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRQLYSALANKCCHVGCTKRSLARFC       SEQ ID NO:15。
2、重组人松弛素800810的转化、筛选、诱导表达
重组人松弛素800810的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800810序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(WT)   QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例16、重组人松弛素800810-1的分子克隆和表达
1、重组人松弛素800810-1表达载体的构建
松弛素800810-1用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
810-1-引物1
CTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGG   SEQ ID NO:97
810-1-引物2
CCAAAGCAGAGTACAAATCTCTCTTAGACCAAGTAG  SEQ ID NO:98
PCR定点突变以松弛素800810载体为模板，突变过程同实施例2。800810-1前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA       SEQ ID NO:99
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRDLYSALANKCCHVGCTKRSLARFC          SEQ ID NO:16。
2、重组人松弛素800810-1的转化、筛选、诱导表达
重组人松弛素800810-1的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800810-1序列如下：
B链(DB14) DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)  DLYSALANKCCHVGCTKRSLARFC          SEQ ID NO:138。
实施例17、重组人松弛素800811的分子克隆和表达
1、重组人松弛素800811表达载体的构建
松弛素800811用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
811-引物1
GTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTG
SEQ ID NO:100
811-引物2
CAATTTGAGCTCTAACCAAATCTCTACCACACAACTTAATAAC
SEQ ID NO:101
PCR定点突变以松弛素800804载体为模板，突变过程同实施例2。800811前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
SEQ ID NO:102
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSKRQLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:17。
2、重组人松弛素800811的转化、筛选、诱导表达
重组人松弛素800811的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800811序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(WT)   QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例18、重组人松弛素800813的分子克隆和表达
1、重组人松弛素800813表达载体的构建
松弛素800813用PCR的方法进行定点突变完成，由Invitrogen公司合成2条 单链DNA片段作为定点突变引物，序列如下：
813-引物1
CTGGTTACGGTTCTAAGAGAGAATTGTACTCTGCTTTGGC
SEQ ID NO:103
813-引物2  GCCAAAGCAGAGTACAATTCTCTCTTAGAACCGTAACCAG
SEQ ID NO:104
PCR定点突变以松弛素800811载体为模板，突变过程同实施例2。800813前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGAGAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
SEQ ID NO:105
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSKRELYSALANKCCHVGCTKRSLARFC         SEQ ID NO:18。
2、重组人松弛素800813的转化、筛选、诱导表达
重组人松弛素800813的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800813序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(EA1)   ELYSALANKCCHVGCTKRSLARFC         SEQ ID NO:136。
实施例19、重组人松弛素800814的分子克隆和表达
1、重组人松弛素800814表达载体的构建
松弛素800814用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
814-引物1CTGGTTACGGTTCTAAGAGAGATTTGTACTCTGCTTTGGC
SEQ ID NO:106
814-引物2
GCCAAAGCAGAGTACAAATCTCTCTTAGAACCGTAACCAG
SEQ ID NO:107
PCR定点突变以松弛素800811载体为模板，突变过程同实施例2。800814前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
SEQ ID NO:108
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSKRDLYSALANKCCHVGCTKRSLARFC       SEQ ID NO:19。
2、重组人松弛素800814的转化、筛选、诱导表达
重组人松弛素800814的转化、筛选、诱导表达同实施例1。
得到的本公开的测试样品重组人松弛素类似物800814序列如下：
B链(DB14) DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)  DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例20、重组人松弛素800816的分子克隆和表达
1、重组人松弛素800816表达载体的构建
松弛素800816用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
816-引物1
ATTAAGTTGTGTGGTAGAAACTTGGTTAGAGCTCAAATTGC
SEQ ID NO:109
816-引物2
GCAATTTGAGCTCTAACCAAGTTTCTACCACACAACTTAAT
SEQ ID NO:110
PCR定点突变以松弛素800804载体为模板，突变过程同实施例2。800816前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAAACTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGACAATTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA
SEQ ID NO:111
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRNLVRAQIAICGMSTWSKRKPTGYGSKRQLYSALANKCCHVGCTKRSLARFC            SEQ ID NO:20。
2、重组人松弛素800816的转化、筛选、诱导表达
重组人松弛素800816的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800816序列如下：
B链(NB14)  DSWMEEVIKLCGRNLVRAQIAICGMSTWS    SEQ ID NO:139
A链(WT)   QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例21、重组人松弛素800847Y的分子克隆和表达
1、重组人松弛素800847Y表达载体的构建
松弛素800847Y用PCR的方法进行定点突变完成，由Invitrogen公司合成2 条单链DNA片段作为定点突变引物，序列如下：
847Y-引物1AAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAA
SEQ ID NO:112
847Y-引物2TTTGAGCTCTAACCAATTCTCTACCACACAACTT
SEQ ID NO:113
PCR定点突变以松弛素800814载体为模板，突变过程同实施例2。800847Y前体DNA序列如下：
GATTCTTGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGAATTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA  SEQ ID NO:114
上述DNA序列编码氨基酸序列如下：
DSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSKRDLYSALANKCCHVGCTKRSLARFC        SEQ ID NO:21
2、重组人松弛素800847Y的转化、筛选、诱导表达
重组人松弛素800847Y的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800847Y序列如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(DA1) DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例22、重组人松弛素800851Y的分子克隆和表达
1、重组人松弛素800851Y表达载体的构建
松弛素800851Y用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
851Y-引物1
GGGGTATCTCTCGAGAAAAGATGGATGGAAGAAGTTATTAAG
SEQ ID NO:115
851Y-引物2
CTTAATAACTTCTTCCATCCATCTTTTCTCGAGAGATACCCC
SEQ ID NO:116
PCR定点突变以松弛素800814载体为模板，突变过程同实施例2。800851Y前体DNA序列如下：
TGGATGGAAGAAGTTATTAAGTTGTGTGGTAGAGATTTGGTTAGAGCTCAAATTGCTATTTGTGGTATGTCTACTTGGTCTAAGAGAAAGCCAACTGGTTACGGTTCTAAGAGAGATTTGTACTCTGCTTTGGCTAACAAGTGTTGTCATGTTGGTTGTACTAAGAGATCTTTGGCTAGATTTTGTTAA   SEQ ID NO:117
上述DNA序列编码氨基酸序列如下：
WMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSKRDLYSALANKCCHVGCTKRSLARFC     SEQ ID NO:22。
2、重组人松弛素800851Y的转化、筛选、诱导表达
重组人松弛素800851Y的转化、筛选、诱导表达同实施例1。
得到的重组人松弛素类似物800851Y序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1)   DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例23、重组人松弛素800828的分子克隆和表达
1、重组人松弛素800828表达载体的构建
经过密码子优化的人松弛素DNA序列，在5’端引入NdeI酶切位点(CATATG)，在3’端引入BamHI酶切位点(GGATCC)。全序列利用Overlap PCR方法合成。由Invitrogen公司合成6条单链DNA片段作为合成引物，序列如下：
828-引物1
CATATGAAGAAAAACATCGCGTTCCTGCTGAAACGTGACTCTTGGATGGA                  SEQ ID NO:118
828-引物2
GCACGAACCAGTTCACGACCGCACAGTTTGATAACTTCTTCCATCCAAGAGTCACGTTT         SEQ ID NO:119
828-引物3
CGTGAACTGGTTCGTGCGCAAATTGCGATCTGCGGTATGTCTACCTGGTCTAAACGTAA         SEQ ID NO:120
828-引物4
CAGCTGACGTTTTTTACGAGAACCGTAACCGGTCGGTTTACGTTTAGACCAGGTAGACA         SEQ ID NO:121
828-引物5
CTCGTAAAAAACGTCAGCTGTACTCTGCGCTGGCGAACAAATGCTGCCACGTTGGTTGC         SEQ ID NO:122
828-引物6
GGATCCTTAGCAGAAACGCGCCAGAGAACGTTTGGTGCAACCAACGTGGCAG                SEQ ID NO:123
松弛素合成采用KOD plus试剂盒(TOYOBO,Cat.KOD-201)，分两步PCR完成。第一步PCR，25μL反应体系：2.5μL 10×KOD buffer，2.5μL 2mM dNTPs，引物1,2,3,4,5,6(10μM)各1μL，0.5μL KOD plus，1μL 25mM MgSO4，12.5μLddH2O。反应程序为94℃5分钟，94℃30秒钟，60℃30秒钟，68℃30秒钟，扩增30个循环后，再68℃30分钟以结束PCR扩增程序。第二步PCR，25μL反应体系：2.5μL 10×KOD buffer，2.5μL 2mM dNTPs，引物1和6(10μM)各1μL，1μL第一轮PCR产物，0.5μL KOD plus，1μL 25mM MgSO4，15.5μL ddH2O。反应程 序为94℃5分钟，94℃30秒钟，68℃60秒钟，扩增30个循环后，再68℃10分钟以结束PCR扩增程序。
将合成的DNA序列和pET9a(Novagen,Cat.69431-3)载体分别进行NdeI/BamHI(Takara,Cat.D1161A/D1010A)双酶切，得到的目的片段经1.2％琼脂糖凝胶回收，用T4DNA连接酶(New England Biolabs,Cat.M0202V)连接，转化DH5α感受态细胞(天根生化，Cat.CB101-02)，挑选阳性克隆由Invitrogen公司作测序分析。800828前体DNA序列如下：
CATATGAAGAAAAACATCGCGTTCCTGCTGAAACGTGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCGCAAATTGCGATCTGCGGTATGTCTACCTGGTCTAAACGTAAACCGACCGGTTACGGTTCTCGTAAAAAACGTCAGCTGTACTCTGCGCTGGCGAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCGCGTTTCTGCTAAGGATCC
SEQ ID NO:124
下划线表示酶切位点。
上述DNA序列编码氨基酸序列如下：
MKKNIAFLLKRDSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSRKKRQLYSALANKCCHVGCTKRSLARFC      SEQ ID NO:23。
2、重组人松弛素800828的小量诱导表达
将测序正确的pET9a-松弛素800828质粒转化BL21(DE3)感受态细胞(天根生化，Cat.CB105-02)，挑取单克隆菌株进行IPTG诱导表达。具体方法如下：从新鲜的平板挑取单克隆菌株，接入10ml含有氨苄青霉素的LB培养基中，37℃震摇培养至OD600值达到0.6。取出部分样品作为未诱导对照及细胞冻存。剩余样品中加入1M IPTG浓储液至终浓度1mM，继续培养4小时。诱导结束后离心收集菌体进行SDS-PAGE电泳分析。
得到的重组人松弛素800828序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例24、重组人松弛素800843的分子克隆和表达
1、重组人松弛素800843表达载体的构建
松弛素800843用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
843-引物1GTTCTCGTAAAAAACGTTGGCTGTACTCTGCGCTG
SEQ ID NO:125
843-引物2CAGCGCAGAGTACAGCCAACGTTTTTTACGAGAAC
SEQ ID NO:126
PCR定点突变以松弛素800828载体为模板，突变过程同实施例18。800843 前体DNA序列如下：
ATGAAGAAAAACATCGCGTTCCTGCTGAAACGTGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCGCAAATTGCGATCTGCGGTATGTCTACCTGGTCTAAACGTAAACCGACCGGTTACGGTTCTCGTAAAAAACGTTGGCTGTACTCTGCGCTGGCGAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCGCGTTTCTGCTAA   SEQ ID NO:127
上述DNA序列编码氨基酸序列如下：
MKKNIAFLLKRDSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSRKKRWLYSALANKCCHVGCTKRSLARFC      SEQ ID NO:24。
2、重组人松弛素800843的小量诱导表达
重组人松弛素800843的小量诱导表达同实施例16。
得到的重组人松弛素类似物800843序列如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WA1) WLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:141。
实施例25、重组人松弛素800847的分子克隆和表达
1、重组人松弛素800847表达载体的构建
松弛素800847用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
847-引物1CAAACTGTGCGGTCGTGAACTGGTTCGTGCGCAAA
SEQ ID NO:128
847-引物2TTTGCGCACGAACCAGTTCACGACCGCACAGTTTG
SEQ ID NO:129
PCR定点突变以松弛素800845载体为模板，突变过程同实施例18。800847前体DNA序列如下：
ATGAAGAAAAACATCGCGTTCCTGCTGAAACGTGACTCTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGAACTGGTTCGTGCGCAAATTGCGATCTGCGGTATGTCTACCTGGTCTAAACGTAAACCGACCGGTTACGGTTCTAAACGTGACCTGTACTCTGCGCTGGCGAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCGCGTTTCTGCTAA      SEQ ID NO:130
上述DNA序列编码氨基酸序列如下：
MKKNIAFLLKRDSWMEEVIKLCGRELVRAQIAICGMSTWSKRKPTGYGSKRDLYSALANKCCHVGCTKRSLARFC     SEQ ID NO:25。
2、重组人松弛素800847的小量诱导表达
重组人松弛素800847的小量诱导表达同实施例16。
得到的重组人松弛素类似物800847序列如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(DA1) DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
实施例26、重组人松弛素800851的分子克隆和表达
1、重组人松弛素800851表达载体的构建
松弛素800851用PCR的方法进行定点突变完成，由Invitrogen公司合成2条单链DNA片段作为定点突变引物，序列如下：
851-引物1CGCGTTCCTGCTGAAACGTTGGATGGAAGAAGTTATC
SEQ ID NO:131
851-引物2GATAACTTCTTCCATCCAACGTTTCAGCAGGAACGCG
SEQ ID NO:132
PCR定点突变以松弛素800845载体为模板，突变过程同实施例18。800851前体DNA序列如下：
ATGAAGAAAAACATCGCGTTCCTGCTGAAACGTTGGATGGAAGAAGTTATCAAACTGTGCGGTCGTGACCTGGTTCGTGCGCAAATTGCGATCTGCGGTATGTCTACCTGGTCTAAACGTAAACCGACCGGTTACGGTTCTAAACGTGACCTGTACTCTGCGCTGGCGAACAAATGCTGCCACGTTGGTTGCACCAAACGTTCTCTGGCGCGTTTCTGCTAA     SEQ ID NO:133
上述DNA序列编码氨基酸序列如下：
MKKNIAFLLKRWMEEVIKLCGRDLVRAQIAICGMSTWSKRKPTGYGSKRDLYSALANKCCHVGCTKRSLARFC     SEQ ID NO:26。
2、重组人松弛素800851的小量诱导表达
重组人松弛素800851的小量诱导表达同实施例16。
得到的重组人松弛素类似物800851序列如下：
B链(DelB1B2,DB14)WMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:140
A链(DA1)DLYSALANKCCHVGCTKRSLARFC           SEQ ID NO:138。
实施例27重组人松弛素800828发酵
1、菌株种子培养
1)菌株：800828表达菌株
2)培养基：
a)LB培养基:Yeast extract 5g/L，yptone 10g/L，NaCl 5g/L，121℃30min高温灭菌，
b)卡那霉素母液：50mg/mL，0.22μm过滤除菌
c)发酵培养基：
Tryptone 20g/L，Yeast extract 10g/L，NaCl 10g/L，Na2HPO4·12H2O 4g/L，KH2PO42g/L，K2HPO42g/L，MgSO41g/L，甘油10g/L，泡敌5mL，全部溶解后，加入5L罐内，121℃灭菌30min。
d)补料培养基：
Tryptone100g/L，Yeast extract 50g/L，甘油500g/L，121℃灭菌30min。
2、发酵过程控制
1)种子活化：将保存于-80℃的种子甘油管室温解冻，取甘油管菌液500μL无菌接入50mL LB培养基，再加入卡那霉素母液5μL，37℃200rpm培养7h。
2)上罐：将上步活化的种子50mL无菌接入3L发酵培养基，再加入卡那霉素30mL，培养条件温度：37℃，空气流量：0.5vvm，罐压：0.04－0.05mpa，DO：30％以上，pH：用氨水维持7.0左右。
3)从发酵4小时左右开始，每间隔1小时左右取样测发酵液的OD600，直至发酵液的OD600≥40时，开始诱导。
4)诱导物：IPTG(1mol/L，用前经0.22μm滤膜无菌过滤)，一次性加入4mL，至IPTG终浓度为1mmol/L，诱导阶段温度控制在37℃，其它条件不变，诱导12小时。
5)诱导结束，放罐，测得此时0D600，发酵液5000rpm离心20min，收集菌体，-80度冰箱中保存。
实施例28重组人松弛素800814发酵
1、菌种：800814表达菌株。
2、5L发酵罐发酵操作流程：
1)菌种活化：
取上述菌种的甘油菌1mL接入100mL BMGY培养基中，30℃，220r/min(rpm)培养20hr。
2)接种：
将活化好的菌种火焰接入3L发酵培养基中(H3PO426.7ml/L，CaSO40.93g/L，K2SO418.2g/L，MgSO4.7H2O 14.9g/L，KOH 4.13g/L，glycerol 40g/L)，并加入0.4％已除菌的PTM1溶液，开始发酵。
3)初始培养阶段：
菌种经过一段适应期(10-12hr)后进入指数生长期，通过不断加大搅拌转速和通气量满足菌体生长所需的DO>30％，转速每次调高50-100rpm。控制发酵温度：37℃，罐压：0.04－0.05Mpa，pH：7.0。
4)甘油补料阶段：
初始培养基中底物消耗完(18-24hr)后，开始流加50％甘油，并且使之处于限制性速率。
5)甲醇诱导阶段：
甘油过渡相菌体生长2至4h后，停止补甘油，维持饥饿状态30min，让甘油彻底耗尽，然后补入甲醇开始诱导。诱导70至96小时后停止发酵，发酵液7000rpm离心，留上清液。
6)目的蛋白表达量：
取1ml发酵上清液用SDS-PAGE考察上清液中目的蛋白的含量，证明有正确的蛋白表达。
实施例29重组人松弛素800828纯化和复性
1、纯化包涵体
1)大肠杆菌湿重100g以300ml 50mM Tris pH8.5，0.2％EDTA悬浮，超声破菌。10000rpm 4℃离心1hr，弃上清。
2)沉淀用300ml 2M Urea,50mM Tris pH8.5，0.2％EDTA充分悬浮。离心弃上清。重复一次。
3)沉淀用200ml 8M Urea,50mM Tris pH8.5，0.2％EDTA充分悬浮。离心弃上清。
4)沉淀用200ml 50mM Tris pH8.5，0.2％EDTA充分悬浮。离心弃上清。沉淀称重。
5)沉淀用6倍量6M GdnCl 50mM Tris pH8.5，0.2％EDTA，10mMDTT充分悬浮，室温溶解2hr。10000rpm 4℃离心1hr，留上清。
6)电泳检测各洗涤组分。
2、复性包涵体
1)获得包涵体以2倍体积50mM Tris pH8.5，0.2％EDTA稀释，混合均匀静置于4℃冰箱，过夜。
2)以RP-UPLC检验复性进程，反应结束后以制备液相纯化，LC-MS检测分子量。
a)RP-UPLC条件：Waters UPLC BioHClass,ACQUITY UPLC BEH 300C4(1.7μm 2.1×100mm)，流动相为：A液0.1％TFA；B液ACN，流速0.3ml/min，线性梯度：0.5min(10B％)至9min(60B％)。
b)制备方法：Water AutoPurifier，Kromasil 10-100-C18，30×250mm；流动相为A液0.1％TFA；B ACN，流速40ml/min，线性梯度2min(25％B)至15min(50％B)。
c)LC-MS方法：Finnigan LCQ Ad，Jupiter C4(5u,300A,250×4.6mm)，流动相为:A液0.2％FA；B:0.18％FA/CAN，线性梯度：0min(5％B)至20min(50B％)。
3、酶切：50mM Tris pH8.5，5mMCa2+,Trpsin 1:1300，CPB1:50，底物浓度1mg/ml，反应温度30℃。以UPLC检测反应进程。反应结束后将体系pH调至3至4终止反应，离心去沉淀。上清经过0.22μm膜过滤后上RP-HPLC制备纯化。反相制备梯度2min(20％B)至15min(45％B)，其他检测条件同上。冻干获得产物(编号828)。以RP-UPLC和IEC-HPLC检验纯度。
1)RP-UPLC条件同上
2)IEC-HPLC：Waters UPLC BioHClass,Protein-Pak Hi Res CM(7μm，4.6×100mm),A液：20mM HEPES pH8.0；B液0.5M NaCl 20mM HEPES pH8.0，线性梯度1min(0％B)至10min(100％B)。
4、环化：产物干粉用1mM HCl溶解，放置于80℃水浴中。UPLC监测反应进程，直至反应结束。检测条件同上。
5、得到本公开的阳性对照，原始序列的样品800828(WT)，以20mM NaAc pH5.0溶解供活性测定。
得到的重组人松弛素800828序列(WT，野生型)如下：
B链(WT)  DSWMEEVIKLCGRELVRAQIAICGMSTWS    SEQ ID NO:134
A链(WT)  QLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:135。
实施例30重组人松弛素800814发酵产物的纯化，鉴定
本公开所设计的系列分子，因为A1和B14引入带不同电荷的氨基酸，发现在酵母中所表达的松弛素前体分子能够在细胞内完成酶切，成熟的松弛素分子分泌到培养基的上清液中。从上期液中可以直接纯化的完整的松弛素分子。步骤如下：
步骤1，上清液过柱纯化
发酵液上清先以Capto MMC(GE17-5318-03)柱使用AKTA purifier纯化。柱子以20mM NaAC pH4平衡，发酵液上清调pH为4后上样。以100mM NaHCO3pH11洗脱，收集洗脱峰。调pH为3后使用RP-HPLC继续纯化。反相制备纯化使用Water AutoPurifier，Kromasil 10-100-C18，30×250mm；流动相为A液0.1％TFA；B ACN，流速40ml/min，线性梯度2min(20％B)至15min(50％B)，收集洗脱峰。冻干获得目的产物，HPLC检测纯度为93％。经LC-MS检测产物分子量为5953.0，与计算分子量5952.9一致，得到的重组人松弛素类似物800814序列如下：
B链(DB14)  DSWMEEVIKLCGRDLVRAQIAICGMSTWS    SEQ ID NO:137
A链(DA1) DLYSALANKCCHVGCTKRSLARFC         SEQ ID NO:138。
步骤2：二硫键鉴定
上述产物溶解于50mM Tris pH8.0，加入胰蛋白酶(Sigma,Cat.T1426)37℃过夜酶切，反应结束后加1M HCl 3μl终止反应。LC-MS检测到片段如下表，证实存在二硫键A24-B23，另两个二硫键由A10、A11、A15和B11构成。
表2.二硫键鉴定结果
Figure PCTCN2014088280-appb-000006
实施例31松弛素800814肽段图谱分析
利用Agilent 1260LC-6530Q-TOF-MS仪器对松弛素800814进行MS分子量测定和序列覆盖率分析。其中，液相条件中的流动相组成：
A相：水(含0.1％甲酸)；B相：乙腈(含0.1％甲酸)，
色谱柱：Poroshell 300SB-C82.1×75mm 5μm，柱温：50℃。洗脱梯度见表3。
表3.洗脱梯度
Figure PCTCN2014088280-appb-000007
质谱条件为：离子源：AJS ESI(+)，离子源参数：Nebulizer 40psig、Drying Gas Temp 325℃、Gas Flow 10l/min，Sheath Gas Temp 350℃,Sheath Gas Flow 12l/min、Vcap 3500V、Fragmentor 200V，扫描范围：m/z 50-3200。
松弛素800814分子量测定：测定分子量为5948.7996Da，和理论值完全吻合，证明蛋白表达正确。
松弛素800814还原肽图分析：利用DTT还原蛋白二硫键对其进行序列覆盖率分析以检测蛋白表达是否正确。条件如下：在0.1mg/mL样品中加入终浓度20mM的DTT孵育2h，然后加入终浓度40mM的IAA避光30min。取处理后样品进行LC-MS检测分析。结果显示，分子序列完全匹配，进一步证明所表达的蛋白分子和预期的一致。
表4.LC-MS检测分析结果
松弛素800814链B DSWMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:137
松弛素800814链A DLYSALANKCCHVGCTKRSLARFC SEQ ID NO:138
实施例31人松弛素类似物800814PEG衍生物制备
1.准确称量10.0mg样品重组人松弛素800814(SEQ ID NO：19，制备于实施例19)于50mL的反应管中，加入5.0mL的pH4.5、4℃的0.1M的CH3COOH/CH3COONa缓冲液溶解样品，重组人松弛素800814终浓度为2.0mg/mL(0.1M冰醋酸/醋酸钠配制：3.7g冰醋酸+3.2g醋酸钠+1000mL水)。
2.准确称量100mg的单甲氧基醛基聚乙二醇(m-PEG-CHO，20kDa)用2.0mL的四氢呋喃/乙腈(1:1)混合溶剂溶解后，加入到上述重组人松弛素800814的 缓冲溶液中(PEG与重组人松弛素800814配比为3:1)。
3.准确称量0.5mg的氰基硼氢化钠(NaCNBH3)溶于1.0mL的pH4.5的缓冲溶液中，再加入到上述重组人松弛素800814的缓冲液中(按照还原剂与修饰剂配比为100:1加入还原剂)。
4.反应混合物在室温(T＝25℃)条件下，反应进行1.0和2.0h，用RP-HPLC检测反应进行，待反应生成物不再进一步增加时，在反应体系中加入1.0mL的10％的甘氨酸溶液，进行10-30min淬灭反应。
5.纯化：采用
Figure PCTCN2014088280-appb-000008
purifier 10高效液相色谱系统，由SP Sepharose Fast Flow阳离子交换介质色谱柱(1.6cm*18cm)，对反应得到的重组人松弛素800814-PEG衍生物(简称PEG-814)进行分离纯化。平衡缓冲溶液用pH＝4.5、浓度为20mmol/L的HAC-NaAC。10倍稀释后上样收集流穿峰，然后用含有1mol/L的NaCl的缓冲液洗脱，收集洗脱峰。流速1.0mL/min，检测波长280nm。得到本公开产品人松弛素类似物衍生物PEG-814。
生物学评价
测试例一：人松弛素及其类似物体外细胞生物活性检测
本公开的人松弛素类似物(800814)与THP-1细胞上的受体结合，诱导THP-1细胞产生cAMP。通过检测cAMP的产生量来测定人松弛素类似物(800814)的体外活性。原始序列的野生型人松弛素类似物800828(WT)(制备于实施例29)用于本测试作为阳性对照。
实验用细胞THP-1(ATCC，货号：TIB-202TM)在37℃,5％CO2悬浮生长，当细胞处于对数生长期时状态较好，可用于实验或传代。每2至3天传代一次，比例1:3至1:4。培养基：RPMI1640+0.05mM b-巯基乙醇+10％FBS。实验当天，对数生长期的THP-1细胞经过离心收集后，重悬在DMEM/F12中，调整细胞密度2×106cells/ml种植于96孔板(每孔50μl),37℃培养箱中孵育30分钟。用稀释液(DEME/F12+0.2％BSA+0.02聚山梨酯80+2μM毛喉素+500μM IBMX)按3倍比例稀释人松弛素类似物。96孔板中加入50μl稀释的药物,混匀2分钟后，37℃培养箱中孵育30分钟。4100rpm/min离心10分钟(4℃)，吸走75μl上清，每孔加入50μl事先预冷的裂解液，操作在冰上进行,在冰上孵育20分钟，可以适当地振荡片刻，然后以4100rpm/min离心10分钟(4℃)。用CAMP ELISA KIT(CELL BIOLABS)进行cAMP的检测。
上述实验中得到的数据用Graphpad Prism进行非线性拟合曲线，得到人松弛素类似物(800814)诱导THP-1细胞产生CAMP的EC50值(ng/ml)。结果见下表5。
表5：人野生型松弛素(800828)及人松弛素类似物(800814)的体外活性检测
松弛素分子 EC50(ng/ml)*
800828(WT) 3.03
800814 1.50
*:EC50为三次实验的平均值
上述结果表明，因为分子结构的改变，800814前体分子不仅能够在细胞内完成酶切(省去表达产物体外酶切纯化步骤)，而且直接分泌到上清中的成熟的松弛素类似物(800814)的体外细胞活性比阳性分子(800828(WT))提高了一倍(EC50从3.05ng/ml提高到1.50ng/ml)。
测试例二：人松弛素及其类似物体内生物活性检测
实验用ICR小鼠(雌性，购自西普尔·必凯实验动物有限公司，许可证：SCXK(沪)2008-0016，18-20g)；饲养环境：SPF级。小鼠购进后，实验室环境饲养2周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。原始序列的野生型人松弛素类似物800828(WT，制备于实施例29)用于本测试作为阳性对照。
实验开始前一周，每只ICR雌鼠皮下注射5μg/100μl/只的β-雌二醇(水不溶，油微溶)，用橄榄油混匀。6天后，将体重低于22g的ICR小鼠去除，其余按体重平均分组。皮下注射松弛素800828(WT)(6μg/100μl/只小鼠)、800814(6μg/100μl/只小鼠)或0.1％苯并红紫4B生理盐水(溶剂对照组)。松弛素800828(WT)和800814均溶于含有0.1％benzopurpurin 4B的生理盐水中。40小时后取耻骨，剥离多余的皮肤和肌肉，显微镜下测量耻骨宽度；以溶剂对照组耻骨宽度均值为1，各给药组与其比值即为相对宽度。结果如下。
表6.耻骨宽度
样品 耻骨相对宽度
溶剂对照 1
阳性800828(WT) 1.24
800814 1.72*
*:800814和阳性分子800828(WT)之间统计差异显著
上述结果表明，阳性分子和800814一样在都能引起小鼠耻骨伸张，但是800814分子药效显著优于阳性分子(1.72vs 1.24，p<0.05)。
测试例三：人松弛素及其类似物大鼠体内半衰期测定
实验用SD大鼠，雌雄各半，共12只，购自西普尔·必凯实验动物有限公司，许可证：SCXK(沪)2008-0016，160-180g，饲养环境：SPF级。取SD大鼠，实验室环境饲养3天，温度20-25℃；湿度40-60％。原始序列的野生型人松弛素类似物800828(WT，制备于实施例29)用于本测试作为阳性对照。
将大鼠随机分3组，每组4只，雌雄个数相同。将对照(生理盐水)、人松弛素(类似物800814,WT 800828)样品分别尾静脉注射大鼠，4只/样品，给药剂量为0.5mg/kg/只。检测样品在注射大鼠后0、0.03、0.08、0.25、0.5、1、1.5、2、4、6、 8小时后眼眶取血。所取血样离心，取上清，-20℃保存，待测。等收集完血液样本后，用人松弛素-2Quantikine ELISA Kit(R&D)检测松弛素的含量。用T1/2计算公式和EXCEL来计算待测药物的T1/2。结果如下：
表7.人松弛素及其类似物大鼠体内半衰期测定
样品 T1/2(小时)
800828(WT) 0.87
800814 0.88
结果表明，本公开分子800814结构的改变不影响其半衰期。
测试例四：人松弛素类似物衍生物PEG-814体内长效活性检测
实验用SHR大鼠，雌性，35周龄，体重370g左右，购自维通利华实验动物有限公司，批号：11400700009329。饲养环境：SPF级。测试样品人松弛素类似物衍生物PEG-814制备于实施例31。
大鼠购进后，5只/笼饲养，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度50-60％。实验开始前，用无创血压计(Softron公司提供，型号BP-98A)测试SHR大鼠基础血压2-3次，选取血压稳定且不低于170mmHg的大鼠按血压随机分为受试药组(PEG-814)和溶剂对照组(生理盐水)，每组各10只。分别尾静脉注射松弛素PEG-814或生理盐水，每次500μl(按30μg/天/大鼠)，每日1次(15：00p.m.)，连续给药6周。每周测量一次血压，记录体重和血压数据。
用excel软件统计各组大鼠体重和血压的变化，受试组体重和血压数据与溶剂组进行t检验，比较给药组和对照组给药前后血压是否存在统计学意义，是否存在显著差异，来评价PEG-814的降血压效果。
表8.PEG-814对SHR大鼠血压和体重的影响
Figure PCTCN2014088280-appb-000009
结果表明，采用一天一次的给药方式，PEG-814能显示明显药效(降低血压)，并显示长效效果；对体重没有影响。常规松弛素(非PEG修饰)需要连续静脉注射给药。

Claims (18)

  1. 一种人松弛素类似物，其包含A链和B链，所述A链和B链的氨基酸序列分别如下式所示：
    A链：A1LYSALANKCCHVGCTKRSLARFC
    B链：DSWMEEVIKLCGRB14LVRAQIAICGMSTWS
    其中A1选自：Q、D、E和W；
    其中B14选自：E、D和N；
    任选地，B1和B2同时缺失；
    当A1为Q时，B14不为E或D。
  2. 如权利要求1所述的人松弛素类似物，其中所述A链和B链的氨基酸序列分别如下式所示：
    A链：A1LYSALANKCCHVGCTKRSLARFC
    B链：DSWMEEVIKLCGRB14LVRAQIAICGMSTWS
    其中A1选自：Q、D、E和W；A1优选为D、E、W，更优选为D；
    其中B14选自：E、D和N；
    当A1为Q时，B14不为E或D。
  3. 如权利要求1所述的人松弛素类似物，其中所述的B14为D。
  4. 如权利要求1所述的人松弛素类似物，其中所述的B链和A链的氨基酸序列选自如下组合：
    B链DSWMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:137
    A链DLYSALANKCCHVGCTKRSLARFC SEQ ID NO:138；
    B链DSWMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:137
    A链QLYSALANKCCHVGCTKRSLARFC SEQ ID NO:135；
    B链DSWMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:137
    A链ELYSALANKCCHVGCTKRSLARFC SEQ ID NO:136；
    B链DSWMEEVIKLCGRNLVRAQIAICGMSTWS SEQ ID NO:139
    A链QLYSALANKCCHVGCTKRSLARFC SEQ ID NO:135；
    B链WMEEVIKLCGRDLVRAQIAICGMSTWS SEQ ID NO:140
    A链DLYSALANKCCHVGCTKRSLARFC SEQ ID NO:138；
    B链DSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO:134
    A链WLYSALANKCCHVGCTKRSLARFC SEQ ID NO:141；
    和
    B链DSWMEEVIKLCGRELVRAQIAICGMSTWS SEQ ID NO:134
    A链DLYSALANKCCHVGCTKRSLARFC SEQ ID NO:138。
  5. 如权利要求1所述的人松弛素类似物，其中所述B链通过连接序列与所述A链连接，所述连接序列的长度为1至15个氨基酸残基，优选为2至8个氨基酸残基。
  6. 如权利要求5所述的人松弛素类似物，其中所述的连接序列的氨基酸序列选自：
    L1：KR，
    L2：KRKPTGYGSRKKR，SEQ ID NO:27，
    L3：KRKPTGYGSRKR，SEQ ID NO:28，
    L4：KRGGGPRR，SEQ ID NO:29，
    L5：KRGGGPKR，SEQ ID NO:30，
    L6：KRKPTGYGSKR,SEQ ID NO:31，和
    L7：KRSLKR SEQ ID NO:32。
  7. 如权利要求1所述的人松弛素类似物，其中所述的人松弛素类似物的N端连接有信号肽序列，所述信号肽序列的长度为4至15个氨基酸残基，优选6至11个氨基酸残基。
  8. 如权利要求7所述的人松弛素类似物，其中所述的信号肽序列选自：
    S1：EEGEPK，SEQ ID NO:33，
    S2：EEGEPKR，SEQ ID NO:34，和
    S3：MKKNIAFLLKR SEQ ID NO:35。
  9. 一种人松弛素类似物衍生物，其中如权利要求1-8任一项所述的人松弛素类似物被PEG分子所修饰；所述的PEG分子的分子量为5-100KDa，优选10-80KDa，更优选15-45KDa，最优选20-40KDa；所述的PEG分子为支链型或直链型。
  10. 一种表达如权利要求1-8任一项所述的人松弛素类似物的表达前体，其氨基酸序列选自SEQ ID NO:1至SEQ ID NO:26的一种或多种。
  11. 一种编码如权利要求10所述的表达前体的多核苷酸。
  12. 一种含有如权利要求11所述的多核苷酸的表达载体。
  13. 一种转化有如权利要求12所述的表达载体的宿主细胞。
  14. 如权利要求13所述的宿主细胞，其中所述的宿主细胞为细菌，优选为大肠杆菌。
  15. 如权利要求13所述的宿主细胞，其中所述的宿主细胞为酵母菌，优选为毕赤酵母。
  16. 一种药物组合物，其含有或由如下组成：
    一种或多种如权利要求1至8任一项所述的人松弛素类似物、和/或一种或多种如权利要求9所述的人松弛素类似物衍生物，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  17. 一种可注射溶液，其含有可溶解形式的如权利要求16所述的药物组合物。
  18. 如权利要求1至8任一项所述的人松弛素类似物、如权利要求9所述的人松弛素类似物衍生物、或如权利要求16所述的药物组合物、或如权利要求17所述的可注射溶液，在制备治疗或预防纤维化疾病或心血管疾病的药物中的用途。
PCT/CN2014/088280 2013-11-07 2014-10-10 人松驰素类似物、其药物组合物及其在医药上的应用 WO2015067113A1 (zh)

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