CN110606883A

CN110606883A - Preparation method of hepatocyte growth factor

Info

Publication number: CN110606883A
Application number: CN201910676310.8A
Authority: CN
Inventors: 游婷婷; 王丁力
Original assignee: Guangzhou Lingteng Biomedical Co ltd
Current assignee: Guangzhou Lingteng Biomedical Co ltd
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2019-12-24

Abstract

The invention provides a preparation method of a hepatocyte growth factor, which comprises the following steps: mixing a zymogen form of hepatocyte growth factor with a hepatocyte growth factor activator; the zymogen-form hepatocyte growth factor is a polypeptide fragment in which an alpha-chain of the hepatocyte growth factor is directly connected with a beta-chain of the hepatocyte growth factor; the hepatocyte growth factor activator is selected from: polypeptide with amino acid sequence shown as SEQ ID NO. 15; or a polypeptide with the amino acid sequence shown as SEQ ID NO.15 and one or more substituted amino acids with unchanged biological activity; or a polypeptide with an amino acid sequence shown as SEQ ID NO. 16; or polypeptide with amino acid sequence shown in SEQ ID NO.16 and one or several amino acid substitutions and unchanged bioactivity. Solves the problems that the mature hepatocyte growth factor is difficult to express, is difficult to produce in large scale and has limited application.

Description

Preparation method of hepatocyte growth factor

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of a hepatocyte growth factor.

Background

Hepatocyte Growth Factor (HGF) is considered to be a growth factor in serum that promotes liver regeneration. HGF is secreted extracellularly as an inactive pro-enzyme and is subsequently cleaved by serine proteases into a mature heterodimer consisting of a disulfide-linked 69kDa α -subunit and a 34kDa β -subunit. HGF affects morphogenesis, cell migration, organ regeneration, tumor invasion in different tissues including liver, lung, kidney, intestine, cardiovascular system.

HGF has also been revealed in recent studies as having potential medicinal value in various diseases, such as liver fibrosis and cirrhosis, kidney fibrosis, lung fibrosis, arteriosclerosis obliterans, and the like. The development of recombinant HGF is therefore of great importance for medical applications. However, the conventional escherichia coli expressed recombinant HGF needs to be subjected to a denaturation and renaturation process during purification, the process is complicated and is not favorable for large-scale production, the modification degree of the yeast expressed recombinant HGF is not completely close to that of the naturally occurring HGF, and the drug effect of the yeast expressed recombinant HGF is unknown. On the other hand, after the HGF recombinant plasmid is injected into a human body, the expression of the HGF recombinant plasmid is different due to individual difference, and the consistency of the drug effect cannot be ensured. And the modification degree of the protein expressed by the mammalian cells is very close to that of the natural form HGF, so that the expression of HGF in the mammalian cells has potential application value.

HGF can be further subdivided into HGF pro form (HGF pro) and HGF mature form (HGF match) depending on whether it is processed to maturity. In practical application, HGF matrix with good biological activity is not easy to directly express in vitro, HGF can be obtained by adopting a method of in vitro recombinant expression of HGFpro, and theoretically, HGF pro is likely to be converted into HGF matrix with biological activity in blood after being injected into a living body, so that the drug effect is exerted. However, due to the complexity of organism metabolism and individual variability, the transformation efficiency varies, and the instability and delay of drug effect may be caused if the drug is used for drug treatment. Moreover, with the development of cell therapy technologies, such as stem cell therapy, CAR-T, which have increasingly high requirements for non-animal origin of the culture medium in view of the high risk of serum, biologically inactive HGF pro cannot be converted into the mature form of HGF under serum-free conditions, and thus will be limited in their applications in these fields.

Disclosure of Invention

Accordingly, the present invention aims to provide a method for producing a hepatocyte growth factor, which enables efficient and stable production of mature forms of HGF having biological activity.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

a method for preparing hepatocyte growth factor, comprising: mixing a zymogen form of hepatocyte growth factor with a hepatocyte growth factor activator;

the zymogen-form hepatocyte growth factor is a polypeptide fragment in which an alpha-chain of the hepatocyte growth factor is directly connected with a beta-chain of the hepatocyte growth factor, and the amino acid sequence of the zymogen-form hepatocyte growth factor is shown as SEQ ID NO. 5;

the hepatocyte growth factor activator is selected from: polypeptide with amino acid sequence shown as SEQ ID NO. 15; or a polypeptide with the amino acid sequence shown as SEQ ID NO.15 and one or more substituted amino acids with unchanged biological activity; or a polypeptide with an amino acid sequence shown as SEQ ID NO. 16; or polypeptide with amino acid sequence shown in SEQ ID NO.16 and one or several amino acid substitutions and unchanged bioactivity.

In some embodiments, the preparation of the zymogen form of hepatocyte growth factor comprises: constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form, transiently transfecting into suspended host cell cells, and culturing; and/or

The preparation method of the hepatocyte growth factor activator comprises the following steps: plasmids expressing hepatocyte growth factor activators are constructed, transiently transfected into suspended host cell cells, and cultured.

In some embodiments, the plasmid expressing the zymogen form of hepatocyte growth factor further comprises a fragment encoding a hepatocyte growth factor signal peptide, wherein the fragment encoding the signal peptide is sequentially linked with the fragment encoding the zymogen form of hepatocyte growth factor; and/or

The plasmid for expressing the hepatocyte growth factor activator also contains a segment for coding TNFR signal peptide, and the TNFR signal peptide is connected with the hepatocyte growth factor activator in sequence.

In some of these embodiments, the sequence of the fragment encoding the signal peptide is set forth in SEQ ID No. 6; and/or

The sequence of the fragment for coding the TNFR signal peptide is shown as SEQ ID NO. 17.

In some of these embodiments, the method of constructing the plasmid for expressing the zymogen form of hepatocyte growth factor comprises the steps of:

using DNA containing HGF gene segment as a template, and amplifying by a primer with a nucleotide sequence shown as SEQ ID NO.9 and a primer with a nucleotide sequence shown as SEQ ID NO.10 to obtain a target segment;

the fragment of interest is ligated to an expression vector.

In some of these embodiments, the expression vector is pMCX.

In some of these embodiments, the fragment of interest is inserted between Not I and BamH I of the expression vector.

In some of these embodiments, the method of mixing the zymogen form of hepatocyte growth factor with an activator of hepatocyte growth factor comprises the steps of:

respectively constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form and a plasmid for expressing an activator of the hepatocyte growth factor;

transfecting the plasmid for expressing the zymogen-form hepatocyte growth factor into a host cell, culturing, separating and purifying to obtain the zymogen-form hepatocyte growth factor;

transfecting the plasmid expressing the hepatocyte growth factor activator into a host cell, culturing, separating and purifying to obtain the hepatocyte growth factor activator;

mixing said zymogen form of hepatocyte growth factor with said hepatocyte growth factor activator.

respectively constructing a plasmid for expressing the hepatocyte growth factor in the zymogen form and a plasmid for expressing an activator of the hepatocyte growth factor, co-transfecting the plasmid for expressing the hepatocyte growth factor in the zymogen form and the plasmid for expressing the activator of the hepatocyte growth factor into a host cell, and culturing.

In some of these embodiments, the pro-enzyme form of hepatocyte growth factor is admixed with the hepatocyte growth factor activator in a mass ratio of 10: 1 to 100: 1.

in some of these embodiments, the host cell is a HEK293 cell.

In some embodiments, the HEK293 cells are cultured by: serum-free culture medium is adopted for suspension culture.

The invention also provides a host cell, which comprises the following specific components:

a host cell transfected or transformed with a plasmid as described above expressing a pro-enzyme form of hepatocyte growth factor and/or a plasmid as described above expressing an activator of hepatocyte growth factor.

Based on the technical scheme, the invention has the following beneficial effects:

the invention mixes the proper hepatocyte growth factor activator with the zymogen-form hepatocyte growth factor with the specific sequence to promote the zymogen-form hepatocyte growth factor to be converted to the mature form, and prepares the mature-form hepatocyte growth factor with the bioactivity, thereby solving the problems that the mature-form hepatocyte growth factor is difficult to express, difficult to produce in large scale and limited in application. In addition, the HGF with the bioactivity and the mature form directly prepared by the invention has wider application prospect and application value, and can be directly used in the fields which need serum-free culture or have high requirements on the animal origin-free culture medium, such as stem cell therapy, CAR-T technology and the like.

Moreover, the invention expresses HGF and HGF activator in zymogen form through a mammal expression platform by constructing an in vitro expression vector, and the two are mixed and then converted into HGF in mature form, compared with the common escherichia coli method, the invention has glycosylation modification which is closer to HGF, does not need in vitro renaturation process after respective expression, has simple steps and is closer to HGF in natural state.

In addition, the mammal cells for expressing the HGF in the zymogen form and the HGF activator are suspension cells, a suspension culture form is adopted, a circular-track shaking bioreactor can be selected during production, the consistency of cell culture conditions in the amplification process from 0.1mL to 2500L can be realized through shaking tracks and parameter control, the condition does not need to be found again for large-scale expression like the traditional mode, and convenience is provided for the subsequent amplification process.

Drawings

FIG. 1 is a flow chart of a study of recombinant expression of hepatocyte growth factor in an example of the invention;

FIG. 2 is a view showing the structure of HGF;

FIG. 3 is a schematic diagram of HGF recombinant plasmid construction;

FIG. 4 is a schematic diagram of HGF-A recombinant plasmid construction;

FIG. 5 shows the sequencing result of HGF-pro gene;

FIG. 6 is a graph of HGF-pro double enzyme digestion gel electrophoresis detection;

FIG. 7 is a graph showing the result of detecting the expression of rhHGF pro in HEK cells by Western Blot;

FIG. 8 shows the results of gel electrophoresis of affinity chromatography purified rhHGF pro;

FIG. 9 shows the result of gel electrophoresis detection of the product of rhHGF pro enzyme digested in vitro by HGF-activator;

FIG. 10 shows the results of the activity detection of rhHGF pro and rhHGF mate expressed by HEK293 cells;

FIG. 11 is a map of a pUC57 plasmid;

FIG. 12 shows the results of gel electrophoresis of HGF-Activator protein samples under reducing conditions;

FIG. 13 shows the results of HGF pro expression using CHO cells;

FIG. 14 shows the change of cell viability in the screening process of co-expressed cell pools of HGF pro and HGF-Activator;

FIG. 15 shows a Western blot to detect co-expression of pro-HGF and HGF-Activator in CHO-DG44 cells.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

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

The invention discloses a preparation method of a hepatocyte growth factor, which is characterized by comprising the following steps: mixing a zymogen form of hepatocyte growth factor with a hepatocyte growth factor activator;

Preferably, the preparation method of the zymogen form of the hepatocyte growth factor comprises: plasmids expressing the hepatocyte growth factor in zymogen form were constructed, transiently transfected into suspended host cells, and cultured.

Preferably, the plasmid for expressing the zymogen form of hepatocyte growth factor further comprises a fragment encoding a hepatocyte growth factor signal peptide, wherein the fragment encoding the signal peptide is connected with the fragment encoding the zymogen form of hepatocyte growth factor in sequence.

Preferably, the nucleotide sequence of the fragment encoding the signal peptide is shown as SEQ ID NO. 6. Alternatively, the nucleotide sequence is a protein that encodes the same sequence as SEQ ID NO.6, but differs from SEQ ID NO.6 due to the degeneracy of the genetic code.

Preferably, the method for constructing the plasmid for expressing the zymogen form of the hepatocyte growth factor comprises the following steps:

the fragment of interest is ligated to an expression vector.

More preferably, the DNA containing the HGF gene fragment is pUC57-HGF, which is: a recombinant plasmid in which an HGF gene fragment was inserted into the plasmid pUC 57.

Specifically, the sequence of the HGF gene fragment is shown as SEQ ID NO. 5.

In other embodiments, other DNAs containing HGF gene fragments can be used as templates for amplification to obtain the hepatocyte growth factor in zymogen form.

Specifically, the target fragment and the expression vector are cut by the same restriction enzyme. Preferably, the expression vector is pMCX, and is subjected to double enzyme digestion by Not I and BamH I, and is connected with a target fragment with Not I and BamH I enzyme digestion sites at the tail end.

In some embodiments, the method for preparing the hepatocyte growth factor activator comprises: plasmids expressing hepatocyte growth factor activators were constructed, transiently transfected into suspended host cells, and cultured.

Preferably, the plasmid for expressing the hepatocyte growth factor activator further comprises a fragment encoding a TNFR signal peptide, and the TNFR signal peptide is connected with the hepatocyte growth factor activator in sequence.

More preferably, the nucleotide sequence of the fragment encoding the TNFR signal peptide is shown in SEQ ID NO. 17. Alternatively, the nucleotide sequence is a protein that encodes the same sequence as SEQ ID NO.17, but differs from SEQ ID NO.17 due to the degeneracy of the genetic code.

Preferably, the host cell is a mammalian cell, more preferably a HEK293 cell. More preferably, the HEK293 cell is a HEK293E cell. Compared with an escherichia coli host, the HEK293 cell has glycosylation modification which is closer to natural HGF, does not need the process of expressing alpha chain and beta chain in vitro renaturation in escherichia coli respectively, and is closer to the natural HGF.

Preferably, the culturing is: serum-free culture medium is adopted for suspension culture. Suspension HEK293 cells are adopted for suspension culture, the growth space of the cells is not limited relative to adherent cells, and large-scale culture and protein expression are facilitated.

A method of mixing the zymogen form of hepatocyte growth factor with an activator of hepatocyte growth factor, comprising the steps of: respectively constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form and a plasmid for expressing an activator of the hepatocyte growth factor; transfecting the plasmid for expressing the zymogen-form hepatocyte growth factor into a host cell, culturing, separating and purifying to obtain the zymogen-form hepatocyte growth factor; transfecting the plasmid expressing the hepatocyte growth factor activator into a host cell, culturing, separating and purifying to obtain the hepatocyte growth factor activator; mixing said zymogen form of hepatocyte growth factor with said hepatocyte growth factor activator.

Preferably, the step of transfecting comprises: sequentially adding the plasmid and the PEI into a host cell, wherein the mass ratio of the plasmid to be transfected to the PEI is 1: (1-3). More preferably 1: 2.

Preferably, the medium used in the transfection is RPMI1640 medium. More preferably, after transfection is completed, Excell293 medium is used for culture. Further preferably, the Excell293 medium further contains 3-4mM of VPA.

In other embodiments, the method of mixing the zymogen form of hepatocyte growth factor with an activator of hepatocyte growth factor comprises the steps of: respectively constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form and a plasmid for expressing an activator of the hepatocyte growth factor, co-transfecting the plasmid for expressing the hepatocyte growth factor in the zymogen form and the plasmid for expressing the activator of the hepatocyte growth factor into a host cell, and culturing. After the two plasmids are cotransfected into a host cell, the two plasmids respectively express a hepatocyte growth factor activator and a hepatocyte growth factor in a zymogen form, and the two plasmids are mixed intracellularly or extracellularly to obtain the mature hepatocyte growth factor.

Preferably, the mass ratio of the plasmid mixture to the PEI upon cotransfection is 1: (1-3). More preferably 1: 2.

A host cell of the invention transfected or transformed with a plasmid as described in any of the above expressing a pro-enzyme form of a hepatocyte growth factor and/or a plasmid as described in any of the above expressing an activator of a hepatocyte growth factor.

The present invention is further illustrated by the following specific examples.

Example 1

This example provides a recombinant expression method of hepatocyte growth factor, and the specific flow chart is shown in fig. 1, and the activity of the recombinant expression method is detected.

1. Protein sequence analysis and primer design

HGF consists of 728 amino acids, including 31 amino acids of signal peptide (SEQ ID NO.2), containing 463 amino acids of alpha chain (SEQ ID NO.3) and 334 amino acids of beta chain (SEQ ID NO. 4). In the HGF protein sequence, there are 5 glycosylation sites in total. Contains 3 glycosylation sites on the alpha chain and 2 glycosylation sites on the beta chain. The sequence of HGF is shown in SEQ ID NO. 1.

SEQ ID NO.1：

MWVTKLLPALLLQHVLLHLLLLPIAIPYAEG(SEQ ID NO.2)QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLR(SEQ ID NO.3)VVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS(SEQ ID NO.4)

HGF consists of 2 subunits, namely an α chain (α -chain) and a β chain (β -chain), which are linked by a disulfide bond to form the mature form of HGF. The structural diagram is shown in fig. 2, and it is clear from the structural diagram analysis that the addition of a histidine tag to HGF β chain does not have a significant effect on purification.

Plasmid pUC57-HGF containing HGF gene fragment is used as a template, and pUC57-HGF is used as a template: a recombinant plasmid in which an HGF gene fragment was inserted into the plasmid pUC 57. A plasmid map of pUC57 is shown in FIG. 11. The sequence of the HGF gene fragment is shown as SEQID NO.5 (the underlined part is a signal peptide):

atgtgggtgaccaaactcctgccagccctgctgctgcagcatgtcctcctgcatctcctcctgctccc catcgccatcccctatgcagagggacaaaggaaaagaagaaatacaattcatgaattcaaaaaatcagcaaagactaccctaatcaaaatagatccagcactgaagataaaaaccaaaaaagtgaatactgcagaccaatgtgctaatagatgtactaggaataaaggacttccattcacttgcaaggcttttgtttttgataaagcaagaaaacaatgcctctggttccccttcaatagcatgtcaagtggagtgaaaaaagaatttggccatgaatttgacctctatgaaaacaaagactacattagaaactgcatcattggtaaaggacgcagctacaagggaacagtatctatcactaagagtggcatcaaatgtcagccctggagttccatgataccacacgaacacagctttttgccttcgagctatcggggtaaagacctacaggaaaactactgtcgaaatcctcgaggggaagaagggggaccctggtgtttcacaagcaatccagaggtacgctacgaagtctgtgacattcctcagtgttcagaagttgaatgcatgacctgcaatggggagagttatcgaggtctcatggatcatacagaatcaggcaagatttgtcagcgctgggatcatcagacaccacaccggcacaaattcttgcctgaaagatatcccgacaagggctttgatgataattattgccgcaatcccgatggccagccgaggccatggtgctatactcttgaccctcacacccgctgggagtactgtgcaattaaaacatgcgctgacaatactatgaatgacactgatgttcctttggaaacaactgaatgcatccaaggtcaaggagaaggctacaggggcactgtcaataccatttggaatggaattccatgtcagcgttgggattctcagtatcctcacgagcatgacatgactcctgaaaatttcaagtgcaaggacctacgagaaaattactgccgaaatccagatgggtctgaatcaccctggtgttttaccactgatccaaacatccgagttggctactgctcccaaattccaaactgtgatatgtcacatggacaagattgttatcgtgggaatggcaaaaattatatgggcaacttatcccaaacaagatctggactaacatgttcaatgtgggacaagaacatggaagacttacatcgtcatatcttctgggaaccagatgcaagtaagctgaatgagaattactgccgaaatccagatgatgatgctcatggaccctggtgctacacgggaaatccactcattccttgggattattgccctatttctcgttgtgaaggtgataccacacctacaatagtcaatttagaccatcccgtaatatcttgtgccaaaacgaaacaattgcgagttgtaaatgggattccaacacgaacaaacataggatggatggttagtttgagatacagaaataaacatatctgcggaggatcattgataaaggagagttgggttcttactgcacgacagtgtttcccttctcgagacttgaaagattatgaagcttggcttggaattcatgatgtccacggaagaggagatgagaaatgcaaacaggttctcaatgtttcccagctggtatatggccctgaaggatcagatctggttttaatgaagcttgccaggcctgctgtcctggatgattttgttagtacgattgatttacctaattatggatgcacaattcctgaaaagaccagttgcagtgtttatggctggggctacactggattgatcaactatgatggcctattacgagtggcacatctctatataatgggaaatgagaaatgcagccagcatcatcgagggaaggtgactctgaatgagtctgaaatatgtgctggggctgaaaagattggatcaggaccatgtgagggggattatggtggcccacttgtttgtgagcaacataaaatgagaatggttcttggtgtcattgttcctggtcgtggatgtgccattccaaatcgtcctggtatttttgtccgagtagcatattatgcaaaatggatacacaaaattattttaacatataaggtaccacagtca

thus, HGF-pro gene amplification primers are designed, and the primer sequences are sent to a primer synthesis company for synthesis. After the primer was synthesized, it was dissolved in water, and the concentration was adjusted to 5. mu.M and stored at-20 ℃.

2. Plasmid construction

A non-mature zymogen form of HGF expression plasmid, namely full-length form of HGF (rhHGF pro) with alpha-chain directly connected with beta-chain, is constructed. The expression vector was pMCX. The recombinant plasmid was named pMCX-proHGF, which contains a signal peptide and His-tag, as shown in FIG. 3.

Wherein, the sequence of rhHGF pro is:

QRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS(SEQ ID NO.6)

using pUC57-HGF as a template, using a forward primer A and a reverse primer B to amplify rhHGF-pro, and purifying to obtain a target fragment. The sequences of primer A and primer B are shown in Table 1.

TABLE 1

Wherein, ATTT and CGC are protection bases, underlined parts are enzyme cutting site sequences, the sequences are used for introducing enzyme cutting sites Not I and BamH I into two ends of a target fragment during amplification so as to facilitate the connection of the target fragment and an expression vector, and the sequences without the enzyme cutting sites are shown in Table 2. In other embodiments, appropriate sequences for introducing cleavage sites may be added at both ends of the table 2, as required by the expression vector and the cleavage site.

TABLE 2

Primer name	Sequence details	SEQ ID NO.
			Primer A	GCCACCATGTGGGTGACCAAAC	9
Primer B	TCAGTGATGATGATGATGATGAGAACCTGACTGTGGTACC	10

And carrying out double enzyme digestion reaction on the purified target fragment and an expression vector pMCX by using Not I/BamH I enzyme, and purifying a product after enzyme digestion. And connecting the target fragment cut by the same restriction enzyme with an expression vector to obtain a recombinant expression plasmid pMCX-proHGF. The recombinant plasmid is transformed into a competent DH5 alpha cell, and a bacterial liquid after transformation is smeared on a plate containing ampicillin to obtain a single colony. 3-5 single colonies are picked up and cultured in 1ml LB culture medium containing ampicillin for 3h at 37 ℃ for bacteria liquid PCR identification experiment. And (4) selecting the PCR positive bacterial colony to be sent to a sequencing company for sequencing, and carrying out sequence comparison after the sequencing company to be tested feeds back a sequencing result.

FIG. 4 shows the sequencing results of the constructed non-mature form of HGF gene. Sequencing results indicated that the immature form of HGF was correctly inserted into the expression vector.

An expression plasmid of HGF Activator (HGF-Activator) was constructed, the sequence of which was derived from the UniProtKB database, protein No. Q04756. The protein with the sequence number of Q04756 consists of a section of signal peptide (SEQ ID NO.11) containing 35 amino acids, a section of leader peptide (SEQ ID NO.12) containing 337 amino acids, a section of short chain (SEQ ID NO.13) containing 35 amino acids and a section of long chain (SEQ ID NO.14) containing 248 amino acids.

The sequence of the protein numbered Q04756 in the UniProtKB database (SEQ ID No.15) is as follows:

MGRWAWVPSPWPPPGLGPFLLLLLLLLLLPRGFQP(SEQ ID NO.11)QPGGNRTESPEPNATATPAIPTILVTSVTSETPATSAPEAEGPQSGGLPPPPRAVPSSSSPQAQALTEDGRPCRFPFRYGGRMLHACTSEGSAHRKWCATTHNYDRDRAWGYCVEATPPPGGPAALDPCASGPCLNGGSCSNTQDPQSYHCSCPRAFTGKDCGTEKCFDETRYEYLEGGDRWARVRQGHVEQCECFGGRTWCEGTRHTACLSSPCLNGGTCHLIVATGTTVCACPPGFAGRLCNIEPDERCFLGNGTGYRGVASTSASGLSCLAWNSDLLYQELHVDSVGAAALLGLGPHAYCRNPDNDERPWCYVVKDSALSWEYCRLEACESLTR(SEQ ID NO.12)VQLSPDLLATLPEPASPGRQACGRRHKKRTFLRPR(SEQ ID NO.13)IIGGSSSLPGSHPWLAAIYIGDSFCAGSLVHTCWVVSAAHCFSHSPPRDSVSVVLGQHFFNRTTDVTQTFGIEKYIPYTLYSVFNPSDHDLVLIRLKKKGDRCATRSQFVQPICLPEPGSTFPAGHKCQIAGWGHLDENVSGYSSSLREALVPLVADHKCSSPEVYGADISPNMLCAGYFDCKSDACQGDSGGPLACEKNGVAYLYGIISWGDGCGRLHKPGVYTRVANYVDWINDRIRPPRRLVAPS(SEQ ID NO.14)

the gene sequence expressing the TNFR signal peptide is connected with the gene sequence expressing the mature segment of protein (373-655 amino acid sequence, SEQ ID NO.16, namely the sequence connecting SEQ ID NO.13 and SEQ ID NO.14) with the sequence number of Q04756 to form a recombinant plasmid named pMCX-HGF-A. The plasmid construction is schematically shown in FIG. 5.

Wherein the nucleotide sequence (SEQ ID NO.17) of the gene for expressing the TNFR signal peptide is as follows:

atgggcctctccaccgtgcctgacctgctgctgccactggtgctcctggagctgttggtgggaatatacccctcaggggttattggac

wherein, the HGF-Activator sequence is from a UniProtKB database, and the sequence number is Q04756. HGF activator consists of a segment of signal peptide containing 35 amino acids, a segment of leader peptide containing 337 amino acids, a segment of short chain containing 35 amino acids and a 248 amino acid long chain.

3. Extraction of recombinant plasmid

Amplifying and culturing the bacterial liquid with correct sequencing, and extracting plasmids by using a Plasmid Midi kit (QIAGEN) according to the following steps:

(1)25mL of overnight shake-cultured bacterial liquid, centrifuging at 4 ℃ at 6000 Xg for 15min, and pouring out culture supernatant;

(2) collecting the thallus and suspending in 4mL of solution P1;

(3) 4mL of solution P2 was added, the tube was inverted 4-6 times, and incubated at room temperature (15-25 ℃) for 5 min.

(4) 4mL of 4 ℃ pre-cooled solution P3 was added, the tube was inverted 4-6 times, and incubated on ice for 15 min.

(5) Centrifuging at 4 deg.C and 20000 Xg for 30 min;

(6) the supernatant was transferred to another clean 50mL tube, centrifuged at 20000 Xg for 15min at 4 ℃. After centrifugation, the supernatant was transferred to another clean 50mL tube;

(7) add 4mL of solution QBT to equilibrate QIAGEN-tip and flow through the column by gravity;

(8) pouring the supernatant of step (6) into QIAGEN-tip and flowing through the column by gravity;

(9) washing QIAGEN-tip 2 times with 10mL of QC solution;

(10) eluting DNA with 5mL of solution QF into a clean 15mL tube;

(11) add 3.5mL of room temperature isopropanol to the eluted DNA and mix well. Centrifuge at 15000 Xg for 30min at 4 ℃. Carefully suck the supernatant out;

(12) the DNA pellet was washed with 2mL of 70% ethanol, centrifuged at 15000 Xg at 4 ℃ for 10 min. Carefully suck the supernatant out;

(13) air drying for 5-10min, dissolving in purified water of certain volume, and storing at-20 deg.C.

4. Recombinant plasmid restriction enzyme identification

Under the aseptic condition, 2 μ g of the extracted plasmid DNA was subjected to an enzyme digestion reaction, and the reaction system is shown in Table 3.

TABLE 3 restriction enzyme reaction System

The enzyme digestion reaction system reacts in water bath at 37 ℃ for 30min, 5 mu L of sample is taken for agarose gel electrophoresis experiment, and the experiment result is recorded by a gel imaging system.

The experimental results are as follows:

FIG. 6 is a schematic view of HGF-pro dicase; wherein, M represents the standard molecular weight, and the 1 lane sample is the extracted pMCX-proHGF plasmid; lane 2 is the sample after digestion with BamHI; lane 3 shows Not I and BamH I samples after digestion. As can be seen from the results, the molecular weights of the target fragments after the digestion are all consistent with those expected, so that all the extracted plasmids are plasmids with the correct insertion of the target fragments.

5. Transient expression of HGF

rhHGF pro is expressed in this protocol by transient transfection of HEK-293E cells. And (3) recovering and culturing the HEK-293E frozen in the liquid nitrogen, and performing transfection operation after the cell growth rate is normal and 3 passages. Before transfection will beCell density was adjusted to 0.5X 10⁶cells/ml, after two medium changes, can be transfected. The plasmid and PEI were premixed in a 1:2 ratio at transfection, with the final plasmid concentration of 1.5. mu.g/10⁶cells, PEI final concentration 3. mu.g/10⁶cells. Cell density was adjusted to 20X 10 at transfection⁶cells/ml, cells were resuspended in fresh RPMI1640 medium. And adding the resuspended cells into the mixture of the premixed plasmids and the PEI, quickly and uniformly mixing, and putting the mixture into a shaker at 37 ℃ for incubation for 3 hours. Fresh Excell293 medium was added and the cell density was diluted to 1.0X 10⁶cells/ml, and 3.75mM sodium Valproate (VPA) were added and the mixture was shake-expressed at 37 ℃ for 5 days.

6. HGF-Activator expression

HGF-A was expressed in this protocol by transient transfection of HEK-293E cells. And (3) recovering and culturing the HEK-293E frozen in the liquid nitrogen, and performing transfection operation after the cell growth rate is normal and 3 passages. Cell density was adjusted to 0.5X 10 before transfection⁶cells/ml, after two medium changes, can be transfected. The plasmid and PEI were premixed in a 1:2 ratio at transfection, with the final plasmid concentration of 1.5. mu.g/10⁶cells, PEI final concentration 3. mu.g/10⁶cells. Cell density was adjusted to 20X 10 at transfection⁶cells/ml, cells were resuspended in fresh RPMI1640 medium. And adding the resuspended cells into the mixture of the premixed plasmids and the PEI, quickly and uniformly mixing, and putting the mixture into a shaker at 37 ℃ for incubation for 3 hours. Fresh Excell293 medium was added and the cell density was diluted to 1.0X 10⁶cells/ml, and 3.75mM VPA was added and shake-expressed at 37 ℃ for 5 days. After 5 days, cell culture supernatants were harvested by centrifugation for enzyme digestion experiments.

7. Expression level detection

HGF expression level detection

Taking a proper amount of HGF sample expressing 3 days, centrifuging at 1300rpm for 5min, and separating culture solution supernatant and cell sediment. After adding 5 Xloading buffer, the mixture was heated at 100 ℃ for 10 min. The prepared protein sample was subjected to SDS-PAGE and subjected to electrophoresis. After the PAGE electrophoresis of the protein sample is finished, the concentrated gel is cut off from the gel plate, the separation gel is put into a 1 XWB membrane transfer buffer for infiltration, and then the protein immunoblotting experiment is carried out. After the membrane transfer, the NC membrane containing the target protein was placed in 5% skim milk and incubated at room temperature for 1 h. The primary antibody, i.e., mouse-derived anti-His antibody, was then incubated overnight at 4 ℃. The next day, the membrane was washed with TBST for 5min and repeated three times. Adding secondary HRP-labelled coat Anti-mouse Ig (H + L) antibody according to the proportion of 1:5000, and incubating for 1H at room temperature. Washing the membrane for 5min by TBST, and repeatedly washing the membrane for three times. Development was performed using the heaven-earth human and Smart ECL kit. Mixing the solution I and the solution II according to a ratio of 1:1, uniformly spreading on a film, and developing in a gel imaging system.

The experimental results are as follows:

FIG. 7 shows the result of rhHGF pro expression detection, wherein lane 1 is a control sample; lane 2 is a sample of rhHGF pro, which is of a size consistent with that expected.

HGF-activator expression level detection:

the detection method is basically the same as the method for detecting the expression level of HGF. The results are shown in FIG. 12. Lane 1 is a HGF-Activator protein sample under reducing conditions.

8. Protein purification experiments

The harvested cell culture fluid was centrifuged at 1500rpm for 5min at 4 ℃ and the cell pellet was discarded, leaving the cell culture supernatant. The culture supernatant containing HGF was filtered using a syringe with a filter membrane diameter of 0.45. mu.m. After filtration, the cell culture supernatant was purified by nickel column affinity chromatography. The purified samples were dialyzed overnight into 1 XPBS, pH7.4, concentrated to a certain concentration in an ultrafiltration tube, sterile filtered and stored at-80 ℃.

The experimental results are as follows:

fig. 8 is the non-mature form of HGF obtained after purification, the size of which is consistent with the expected. Wherein, lane 1 is the penetrating fluid after the cell culture fluid is combined with the filler; lane 2 is the breakthrough fluid when the buffer washes the packing; lane 3 is the protein sample eluted with 100mM imidazole; lane 4 is the protein sample eluted with 200mM imidazole; lane 5 is a protein sample eluted with 250mM imidazole.

9. In vitro enzyme digestion experiment of rhHGF pro

First, HGF-Activator, a protease that recombinantly processes HGF, is expressed in HEK293 cells. 5ml of HGF-Activator-containing cell culture medium was concentrated to 80. mu.L for use.

25 μ g of 0.5mg/ml rhHGF pro subjected to nickel column affinity chromatography was divided into 5 parts on average, each containing 5 μ g of rhHGF pro. Adding 4 mul, 2 mul, 1 mul, 0.5 mul and 0.25 mul of concentrated HGF-Activator into each part of rhHGF pro, supplementing buffer solution to 20 mul, and reacting at room temperature overnight. A10. mu.l sample was taken the following day for SDS-PAGE detection. The results are shown in FIG. 9. It can be seen that rhHGF pro can be converted into mature form of HGF after incubation with HGF-Activator, i.e., the figure has separate HGF-alpha-chain and beta-chain. It was thus demonstrated that rhHGF obtained in this project is in a zymogen form and can be converted into the mature form by the action of a protease.

10. HGF Activity assay

Human normal hepatocyte-L02 cells were recovered from liquid nitrogen tanks and grown in RPMI1640 (10% FBS). After 3 passages at 3X 10⁴Cells were plated at density of 100. mu.l/well in 96-well plates in 6 replicate wells. After 24h, cell exchange was performed. Adding fresh culture medium containing rhHGF match and rhHGF pro with concentration of 0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml under aseptic condition. After incubation at 37 ℃ for 3 days, MTT was added and incubated for 5h, and the culture medium was aspirated. Adding a proper amount of DMSO into each well, placing on a shaking bed, oscillating at low speed for 10min, and measuring the absorbance of each well at 570 nm.

The experimental results are as follows:

FIG. 10 shows the result of HGF activity assay, which is performed by adding rhHGF mate and rhHGF pro to L02 cells, respectively, and detecting proliferation by MTT method. From the results, it is clear that both rhHGF mate and rhHGF pro can promote the proliferation of L02 cells. The reason is that rhHGF pro is an HGF in zymogen form, has no biological activity, but after being added into cells cultured in a serum-containing culture medium, the serum contains an enzyme capable of converting rhHGF pro into rhHGF match, so that the biological activity can be exerted. However, since it requires a transformation process, especially in a low concentration state, the effect of promoting proliferation of L02 cells is worse than that of rhHGF growth, which means that it needs to spend a certain transformation time before exerting its biological activity, and there is some inactive rhHGF pro which is not transformed into rhHGF growth, so the biological activity is slightly lower, and the rate of cell proliferation is slower in vitro experiments, and further, in practical application in clinic, the transformation efficiency is more different due to individual difference, and the instability and delay of drug effect caused by drug therapy will be more obvious, and its application is limited. The rhHGF mate prepared by the invention is mature HGF, can directly and quickly exert the bioactivity without consuming conversion time when being directly applied, and has the advantages of good effect, quick response and stable drug effect.

Example 2

The differences from example 1 are: the CHO-DG44 cells used for expressing HGF pro and HGF Activator in the embodiment are as follows: the CHO-DG44 cells frozen in liquid nitrogen were recovered and cultured, and transfection operation was performed after 2 passages at normal cell growth rate. Cell density was adjusted to 1.5X 10 the day before transfection⁶cells/ml, the next day cell density expected to be as long as 4X 10⁶cells/ml, cell density adjusted to 5X 10⁶cells/ml, and fresh proCH5 medium was replaced. The transfection reagent used in the transfection experiment was Polyethyleneimine (PEI), with a DNA to PEI ratio of 1: 5. The final DNA concentration was 0.6. mu.g/10⁶cells/ml, final PEI concentration 3. mu.g/10⁶cells/ml. And sequentially adding the DNA and the PEI into the resuspended CHO-DGG cells, quickly mixing uniformly, and then putting into a shaker at 31 ℃ for expression. After 3 days of expression, a proper amount of cell samples are taken for SDS-PAGE and Western blot detection. After 5 days of transfection, transfected cells were harvested for detection and purification.

The results of CHO expression of HGF pro are shown in FIG. 13. Lane 1 is a sample of HGF pro protein under non-reducing conditions.

The results were:

when HGF pro is expressed using CHO-DG44 cells, HGF pro is processed into two protein forms with similar molecular weight sizes, probably due to the characteristics of the host cell itself, see FIG. 13 results. When the HEK cells are used for expressing the HGF pro, the HGF pro only contains a single protein form, so that the HGF pro generated by the HEK cells is single in form, and the method is more suitable for large-scale production and application.

Example 3

HGF pro and HGF Activator are co-expressed in this protocol by stably transfecting CHO-DG44 cells. The CHO-DG44 frozen in liquid nitrogen is recovered and cultured, and transfection operation can be carried out after the cell growth rate is normal and 3 passages. Cell density was adjusted to 2.0X 10 the day before transfection⁶cells/ml, the next day cell density was 4.5-4.8X 10⁶cells/ml. At the time of transfection, the cell density was adjusted to 3.0X 10⁶cells/ml, and fresh proCH5 medium was replaced. The transfection reagent used in the transfection experiment was Polyethyleneimine (PEI), and the ratio of total DNA to PEI was 1: 5. The final concentration of plasmid DNA was 1.5. mu.g/10⁶cells, PEI final concentration 7.5. mu.g/10⁶cells. In the transfection experiment, plasmid DNA including p2MPT-pro-HGF containing target genes and pMPTN-HGF-Activator is added, and the mass ratio is 9: 1. Meanwhile, the recombinant plasmid pMPT-PBase-ori containing transposase gene is added, and the addition amount is 1/10 of the total amount of DNA. DNA and PEI were added sequentially to fresh medium resuspended CHO-DG44 cells according to the following table, mixed rapidly and then transfected in a 37 ℃ shaker.

HGF pro and HGF-Activator co-expression cell pool screening

After recombinant p2MPT-pro-HGF and pMPTN-HGF-Activator transfect CHO-DG44 cells for 72h, the cell density is adjusted to 0.5 x 10⁶cells/ml, puromycin to 10. mu.g/ml, and shake-culturing at 37 ℃. After that, the liquid exchange operation was performed every 2 days, and the cell density was adjusted to 0.5X 10⁶cells/ml and puromycin concentration was added to 10. mu.g/ml. After 10 days of screening, a positive cell pool can be obtained. The cell viability rate varied during the screening process as shown in FIG. 14. According to the results, the cell survival rate of the cell pool after 10 days of screening reaches 96%.

Cell pool expression

Is obtainingAfter obtaining the positive cell pool, the cell density was adjusted to 2.0X 10⁶cells/ml, culture volume adjusted to 10 ml/tube, and added sodium butyrate to 1mM, placed at 31 ℃ for 5 days of shake culture. After completion of expression, the expression supernatant was harvested by centrifugation at 1500rpm for 5 min. An appropriate amount of sample was taken for Western Blot experiment. From the results (see FIG. 15), Lane 1 is HGFfeature (protein molecular weight about 85kD), and Lane 2 is treated with a reducing agent to show a clear beta chain (protein molecular weight about 37 kD). Therefore, HGF match can be obtained after HGFpro and HGF-Activator are co-expressed. Lane 1 is a sample of HGF pro and HGF-Activator co-expressed protein under non-reducing conditions; lane 2 is a sample of HGF pro and HGF-Activator co-expressed protein under reducing conditions.

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

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

Sequence listing

<110> Fushan Hanteng Biotech Co., Ltd

Guangzhou Hanteng Biotech Co., Ltd

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Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr

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Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys

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Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe

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Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys

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Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His

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Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr

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Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser

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Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu

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Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp

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His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro

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His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp

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Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp

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Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met

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Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp

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Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp

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Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr

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Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys

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

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Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp

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Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu

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Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn

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Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu

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Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr

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His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn

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Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr

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Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met

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Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr

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Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys

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Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu

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Asp Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr

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Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys

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Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp

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

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Asp Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile

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Pro Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu

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Asn Glu Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu

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atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

agctttttgc cttcgagcta tcggggtaaa gacctacagg aaaactactg tcgaaatcct 540

cgaggggaag aagggggacc ctggtgtttc acaagcaatc cagaggtacg ctacgaagtc 600

tgtgacattc ctcagtgttc agaagttgaa tgcatgacct gcaatgggga gagttatcga 660

ggtctcatgg atcatacaga atcaggcaag atttgtcagc gctgggatca tcagacacca 720

caccggcaca aattcttgcc tgaaagatat cccgacaagg gctttgatga taattattgc 780

cgcaatcccg atggccagcc gaggccatgg tgctatactc ttgaccctca cacccgctgg 840

gagtactgtg caattaaaac atgcgctgac aatactatga atgacactga tgttcctttg 900

gaaacaactg aatgcatcca aggtcaagga gaaggctaca ggggcactgt caataccatt 960

tggaatggaa ttccatgtca gcgttgggat tctcagtatc ctcacgagca tgacatgact 1020

cctgaaaatt tcaagtgcaa ggacctacga gaaaattact gccgaaatcc agatgggtct 1080

gaatcaccct ggtgttttac cactgatcca aacatccgag ttggctactg ctcccaaatt 1140

ccaaactgtg atatgtcaca tggacaagat tgttatcgtg ggaatggcaa aaattatatg 1200

ggcaacttat cccaaacaag atctggacta acatgttcaa tgtgggacaa gaacatggaa 1260

gacttacatc gtcatatctt ctgggaacca gatgcaagta agctgaatga gaattactgc 1320

cgaaatccag atgatgatgc tcatggaccc tggtgctaca cgggaaatcc actcattcct 1380

tgggattatt gccctatttc tcgttgtgaa ggtgatacca cacctacaat agtcaattta 1440

gaccatcccg taatatcttg tgccaaaacg aaacaattgc gagttgtaaa tgggattcca 1500

acacgaacaa acataggatg gatggttagt ttgagataca gaaataaaca tatctgcgga 1560

ggatcattga taaaggagag ttgggttctt actgcacgac agtgtttccc ttctcgagac 1620

ttgaaagatt atgaagcttg gcttggaatt catgatgtcc acggaagagg agatgagaaa 1680

tgcaaacagg ttctcaatgt ttcccagctg gtatatggcc ctgaaggatc agatctggtt 1740

ttaatgaagc ttgccaggcc tgctgtcctg gatgattttg ttagtacgat tgatttacct 1800

aattatggat gcacaattcc tgaaaagacc agttgcagtg tttatggctg gggctacact 1860

ggattgatca actatgatgg cctattacga gtggcacatc tctatataat gggaaatgag 1920

aaatgcagcc agcatcatcg agggaaggtg actctgaatg agtctgaaat atgtgctggg 1980

gctgaaaaga ttggatcagg accatgtgag ggggattatg gtggcccact tgtttgtgag 2040

caacataaaa tgagaatggt tcttggtgtc attgttcctg gtcgtggatg tgccattcca 2100

aatcgtcctg gtatttttgt ccgagtagca tattatgcaa aatggataca caaaattatt 2160

ttaacatata aggtaccaca gtca 2184

<210> 6

<211> 697

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys

1 5 10 15

Thr Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys

20 25 30

Val Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly

35 40 45

Leu Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln

50 55 60

Cys Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu

65 70 75 80

Phe Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn

85 90 95

Cys Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr

100 105 110

Lys Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu

115 120 125

His Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn

130 135 140

Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr

145 150 155 160

Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser

165 170 175

Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met

180 185 190

Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr

195 200 205

Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe

210 215 220

Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys

225 230 235 240

Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr

245 250 255

Cys Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr

260 265 270

Glu Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr

275 280 285

Ile Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His

290 295 300

Glu His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu

305 310 315 320

Asn Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr

325 330 335

Thr Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys

340 345 350

Asp Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr

355 360 365

Met Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp

370 375 380

Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp

385 390 395 400

Ala Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala

405 410 415

His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr

420 425 430

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

435 440 445

Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val

450 455 460

Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu

465 470 475 480

Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser

485 490 495

Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp

500 505 510

Tyr Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu

515 520 525

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

530 535 540

Gly Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp

545 550 555 560

Asp Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro

565 570 575

Glu Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile

580 585 590

Asn Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn

595 600 605

Glu Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser

610 615 620

Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly

625 630 635 640

Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val

645 650 655

Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro

660 665 670

Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile

675 680 685

Ile Leu Thr Tyr Lys Val Pro Gln Ser

690 695

<210> 8

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atttgcggcc gcgccaccat gtgggtgacc aaac 34

<210> 8

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgcggatcct cagtgatgat gatgatgatg agaacctgac tgtggtacc 49

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccaccatgt gggtgaccaa ac 22

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcagtgatga tgatgatgat gagaacctga ctgtggtacc 40

<210> 11

<211> 35

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Gly Arg Trp Ala Trp Val Pro Ser Pro Trp Pro Pro Pro Gly Leu

1 5 10 15

Gly Pro Phe Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Arg Gly

20 25 30

Phe Gln Pro

35

<210> 12

<211> 337

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Pro Gly Gly Asn Arg Thr Glu Ser Pro Glu Pro Asn Ala Thr Ala

1 5 10 15

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

20 25 30

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

35 40 45

Pro Pro Pro Pro Arg Ala Val Pro Ser Ser Ser Ser Pro Gln Ala Gln

50 55 60

Ala Leu Thr Glu Asp Gly Arg Pro Cys Arg Phe Pro Phe Arg Tyr Gly

65 70 75 80

Gly Arg Met Leu His Ala Cys Thr Ser Glu Gly Ser Ala His Arg Lys

85 90 95

Trp Cys Ala Thr Thr His Asn Tyr Asp Arg Asp Arg Ala Trp Gly Tyr

100 105 110

Cys Val Glu Ala Thr Pro Pro Pro Gly Gly Pro Ala Ala Leu Asp Pro

115 120 125

Cys Ala Ser Gly Pro Cys Leu Asn Gly Gly Ser Cys Ser Asn Thr Gln

130 135 140

Asp Pro Gln Ser Tyr His Cys Ser Cys Pro Arg Ala Phe Thr Gly Lys

145 150 155 160

Asp Cys Gly Thr Glu Lys Cys Phe Asp Glu Thr Arg Tyr Glu Tyr Leu

165 170 175

Glu Gly Gly Asp Arg Trp Ala Arg Val Arg Gln Gly His Val Glu Gln

180 185 190

Cys Glu Cys Phe Gly Gly Arg Thr Trp Cys Glu Gly Thr Arg His Thr

195 200 205

Ala Cys Leu Ser Ser Pro Cys Leu Asn Gly Gly Thr Cys His Leu Ile

210 215 220

Val Ala Thr Gly Thr Thr Val Cys Ala Cys Pro Pro Gly Phe Ala Gly

225 230 235 240

Arg Leu Cys Asn Ile Glu Pro Asp Glu Arg Cys Phe Leu Gly Asn Gly

245 250 255

Thr Gly Tyr Arg Gly Val Ala Ser Thr Ser Ala Ser Gly Leu Ser Cys

260 265 270

Leu Ala Trp Asn Ser Asp Leu Leu Tyr Gln Glu Leu His Val Asp Ser

275 280 285

Val Gly Ala Ala Ala Leu Leu Gly Leu Gly Pro His Ala Tyr Cys Arg

290 295 300

Asn Pro Asp Asn Asp Glu Arg Pro Trp Cys Tyr Val Val Lys Asp Ser

305 310 315 320

Ala Leu Ser Trp Glu Tyr Cys Arg Leu Glu Ala Cys Glu Ser Leu Thr

325 330 335

Arg

<210> 13

<211> 35

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Val Gln Leu Ser Pro Asp Leu Leu Ala Thr Leu Pro Glu Pro Ala Ser

1 5 10 15

Pro Gly Arg Gln Ala Cys Gly Arg Arg His Lys Lys Arg Thr Phe Leu

20 25 30

Arg Pro Arg

35

<210> 14

<211> 248

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ile Ile Gly Gly Ser Ser Ser Leu Pro Gly Ser His Pro Trp Leu Ala

1 5 10 15

Ala Ile Tyr Ile Gly Asp Ser Phe Cys Ala Gly Ser Leu Val His Thr

20 25 30

Cys Trp Val Val Ser Ala Ala His Cys Phe Ser His Ser Pro Pro Arg

35 40 45

Asp Ser Val Ser Val Val Leu Gly Gln His Phe Phe Asn Arg Thr Thr

50 55 60

Asp Val Thr Gln Thr Phe Gly Ile Glu Lys Tyr Ile Pro Tyr Thr Leu

65 70 75 80

Tyr Ser Val Phe Asn Pro Ser Asp His Asp Leu Val Leu Ile Arg Leu

85 90 95

Lys Lys Lys Gly Asp Arg Cys Ala Thr Arg Ser Gln Phe Val Gln Pro

100 105 110

Ile Cys Leu Pro Glu Pro Gly Ser Thr Phe Pro Ala Gly His Lys Cys

115 120 125

Gln Ile Ala Gly Trp Gly His Leu Asp Glu Asn Val Ser Gly Tyr Ser

130 135 140

Ser Ser Leu Arg Glu Ala Leu Val Pro Leu Val Ala Asp His Lys Cys

145 150 155 160

Ser Ser Pro Glu Val Tyr Gly Ala Asp Ile Ser Pro Asn Met Leu Cys

165 170 175

Ala Gly Tyr Phe Asp Cys Lys Ser Asp Ala Cys Gln Gly Asp Ser Gly

180 185 190

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

195 200 205

Ile Ser Trp Gly Asp Gly Cys Gly Arg Leu His Lys Pro Gly Val Tyr

210 215 220

Thr Arg Val Ala Asn Tyr Val Asp Trp Ile Asn Asp Arg Ile Arg Pro

225 230 235 240

Pro Arg Arg Leu Val Ala Pro Ser

245

<210> 15

<211> 655

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Gly Arg Trp Ala Trp Val Pro Ser Pro Trp Pro Pro Pro Gly Leu

1 5 10 15

Gly Pro Phe Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Arg Gly

20 25 30

Phe Gln Pro Gln Pro Gly Gly Asn Arg Thr Glu Ser Pro Glu Pro Asn

35 40 45

Ala Thr Ala Thr Pro Ala Ile Pro Thr Ile Leu Val Thr Ser Val Thr

50 55 60

Ser Glu Thr Pro Ala Thr Ser Ala Pro Glu Ala Glu Gly Pro Gln Ser

65 70 75 80

Gly Gly Leu Pro Pro Pro Pro Arg Ala Val Pro Ser Ser Ser Ser Pro

85 90 95

Gln Ala Gln Ala Leu Thr Glu Asp Gly Arg Pro Cys Arg Phe Pro Phe

100 105 110

Arg Tyr Gly Gly Arg Met Leu His Ala Cys Thr Ser Glu Gly Ser Ala

115 120 125

His Arg Lys Trp Cys Ala Thr Thr His Asn Tyr Asp Arg Asp Arg Ala

130 135 140

Trp Gly Tyr Cys Val Glu Ala Thr Pro Pro Pro Gly Gly Pro Ala Ala

145 150 155 160

Leu Asp Pro Cys Ala Ser Gly Pro Cys Leu Asn Gly Gly Ser Cys Ser

165 170 175

Asn Thr Gln Asp Pro Gln Ser Tyr His Cys Ser Cys Pro Arg Ala Phe

180 185 190

Thr Gly Lys Asp Cys Gly Thr Glu Lys Cys Phe Asp Glu Thr Arg Tyr

195 200 205

Glu Tyr Leu Glu Gly Gly Asp Arg Trp Ala Arg Val Arg Gln Gly His

210 215 220

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

225 230 235 240

Arg His Thr Ala Cys Leu Ser Ser Pro Cys Leu Asn Gly Gly Thr Cys

245 250 255

His Leu Ile Val Ala Thr Gly Thr Thr Val Cys Ala Cys Pro Pro Gly

260 265 270

Phe Ala Gly Arg Leu Cys Asn Ile Glu Pro Asp Glu Arg Cys Phe Leu

275 280 285

Gly Asn Gly Thr Gly Tyr Arg Gly Val Ala Ser Thr Ser Ala Ser Gly

290 295 300

Leu Ser Cys Leu Ala Trp Asn Ser Asp Leu Leu Tyr Gln Glu Leu His

305 310 315 320

Val Asp Ser Val Gly Ala Ala Ala Leu Leu Gly Leu Gly Pro His Ala

325 330 335

Tyr Cys Arg Asn Pro Asp Asn Asp Glu Arg Pro Trp Cys Tyr Val Val

340 345 350

Lys Asp Ser Ala Leu Ser Trp Glu Tyr Cys Arg Leu Glu Ala Cys Glu

355 360 365

Ser Leu Thr Arg Val Gln Leu Ser Pro Asp Leu Leu Ala Thr Leu Pro

370 375 380

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

385 390 395 400

Arg Thr Phe Leu Arg Pro Arg Ile Ile Gly Gly Ser Ser Ser Leu Pro

405 410 415

Gly Ser His Pro Trp Leu Ala Ala Ile Tyr Ile Gly Asp Ser Phe Cys

420 425 430

Ala Gly Ser Leu Val His Thr Cys Trp Val Val Ser Ala Ala His Cys

435 440 445

Phe Ser His Ser Pro Pro Arg Asp Ser Val Ser Val Val Leu Gly Gln

450 455 460

His Phe Phe Asn Arg Thr Thr Asp Val Thr Gln Thr Phe Gly Ile Glu

465 470 475 480

Lys Tyr Ile Pro Tyr Thr Leu Tyr Ser Val Phe Asn Pro Ser Asp His

485 490 495

Asp Leu Val Leu Ile Arg Leu Lys Lys Lys Gly Asp Arg Cys Ala Thr

500 505 510

Arg Ser Gln Phe Val Gln Pro Ile Cys Leu Pro Glu Pro Gly Ser Thr

515 520 525

Phe Pro Ala Gly His Lys Cys Gln Ile Ala Gly Trp Gly His Leu Asp

530 535 540

Glu Asn Val Ser Gly Tyr Ser Ser Ser Leu Arg Glu Ala Leu Val Pro

545 550 555 560

Leu Val Ala Asp His Lys Cys Ser Ser Pro Glu Val Tyr Gly Ala Asp

565 570 575

Ile Ser Pro Asn Met Leu Cys Ala Gly Tyr Phe Asp Cys Lys Ser Asp

580 585 590

Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Lys Asn Gly

595 600 605

Val Ala Tyr Leu Tyr Gly Ile Ile Ser Trp Gly Asp Gly Cys Gly Arg

610 615 620

Leu His Lys Pro Gly Val Tyr Thr Arg Val Ala Asn Tyr Val Asp Trp

625 630 635 640

Ile Asn Asp Arg Ile Arg Pro Pro Arg Arg Leu Val Ala Pro Ser

645 650 655

<210> 16

<211> 283

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Val Gln Leu Ser Pro Asp Leu Leu Ala Thr Leu Pro Glu Pro Ala Ser

1 5 10 15

Pro Gly Arg Gln Ala Cys Gly Arg Arg His Lys Lys Arg Thr Phe Leu

20 25 30

Arg Pro Arg Ile Ile Gly Gly Ser Ser Ser Leu Pro Gly Ser His Pro

35 40 45

Trp Leu Ala Ala Ile Tyr Ile Gly Asp Ser Phe Cys Ala Gly Ser Leu

50 55 60

Val His Thr Cys Trp Val Val Ser Ala Ala His Cys Phe Ser His Ser

65 70 75 80

Pro Pro Arg Asp Ser Val Ser Val Val Leu Gly Gln His Phe Phe Asn

85 90 95

Arg Thr Thr Asp Val Thr Gln Thr Phe Gly Ile Glu Lys Tyr Ile Pro

100 105 110

Tyr Thr Leu Tyr Ser Val Phe Asn Pro Ser Asp His Asp Leu Val Leu

115 120 125

Ile Arg Leu Lys Lys Lys Gly Asp Arg Cys Ala Thr Arg Ser Gln Phe

130 135 140

Val Gln Pro Ile Cys Leu Pro Glu Pro Gly Ser Thr Phe Pro Ala Gly

145 150 155 160

His Lys Cys Gln Ile Ala Gly Trp Gly His Leu Asp Glu Asn Val Ser

165 170 175

Gly Tyr Ser Ser Ser Leu Arg Glu Ala Leu Val Pro Leu Val Ala Asp

180 185 190

His Lys Cys Ser Ser Pro Glu Val Tyr Gly Ala Asp Ile Ser Pro Asn

195 200 205

Met Leu Cys Ala Gly Tyr Phe Asp Cys Lys Ser Asp Ala Cys Gln Gly

210 215 220

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

225 230 235 240

Tyr Gly Ile Ile Ser Trp Gly Asp Gly Cys Gly Arg Leu His Lys Pro

245 250 255

Gly Val Tyr Thr Arg Val Ala Asn Tyr Val Asp Trp Ile Asn Asp Arg

260 265 270

Ile Arg Pro Pro Arg Arg Leu Val Ala Pro Ser

275 280

<210> 17

<211> 88

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

atgggcctct ccaccgtgcc tgacctgctg ctgccactgg tgctcctgga gctgttggtg 60

ggaatatacc cctcaggggt tattggac 88

Claims

1. A method for preparing hepatocyte growth factor, comprising: mixing a zymogen form of hepatocyte growth factor with a hepatocyte growth factor activator;

the zymogen-form hepatocyte growth factor is a polypeptide fragment formed by connecting an alpha-chain of the hepatocyte growth factor with a beta-chain of the hepatocyte growth factor, and the amino acid sequence of the zymogen-form hepatocyte growth factor is shown as SEQ ID NO. 6;

2. The method of claim 1, wherein the zymogen form of hepatocyte growth factor is prepared by a method comprising: constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form, transiently transfecting into suspended host cells, and culturing; and/or

The preparation method of the hepatocyte growth factor activator comprises the following steps: plasmids expressing hepatocyte growth factor activators were constructed, transiently transfected into suspended host cells, and cultured.

3. The method of claim 2, wherein the plasmid expressing the pro-enzyme form of hepatocyte growth factor further comprises a fragment encoding a hepatocyte growth factor signal peptide, wherein the fragment encoding the signal peptide is sequentially linked to the fragment encoding the pro-enzyme form of hepatocyte growth factor; preferably, the sequence of the fragment encoding the signal peptide is shown in SEQ ID NO. 6.

4. The method of claim 2, wherein the hepatocyte growth factor activator-expressing plasmid further comprises a fragment encoding a TNFR signal peptide linked to the hepatocyte growth factor activator-expressing plasmid in sequence; preferably, the sequence of the fragment encoding the TNFR signal peptide is shown in SEQ ID NO. 17.

5. The method of claim 2, wherein the plasmid expressing the pro-enzyme form of the hepatocyte growth factor is constructed by the method comprising the steps of:

the fragment of interest is ligated to an expression vector.

6. The method of claim 5, wherein the expression vector is pMCX.

7. The method of claim 6, wherein the desired fragment is inserted between Not I and BamH I of the expression vector.

8. The method of claim 1, wherein the admixture of the zymogen form of hepatocyte growth factor with an activator of hepatocyte growth factor comprises the steps of:

9. The method of claim 1, wherein the admixture of the zymogen form of hepatocyte growth factor with an activator of hepatocyte growth factor comprises the steps of:

respectively constructing a plasmid for expressing the hepatocyte growth factor in a zymogen form and a plasmid for expressing an activator of the hepatocyte growth factor, co-transfecting the plasmid for expressing the hepatocyte growth factor in the zymogen form and the plasmid for expressing the activator of the hepatocyte growth factor into a host cell, and culturing.

10. The method of any one of claims 2-9, wherein the pro-enzyme form of hepatocyte growth factor is mixed with the hepatocyte growth factor activator at a mass ratio of: 10: 1 to 100: 1.

11. the method of any one of claims 2-9, wherein the host cell is a HEK293 cell.

12. The method for preparing hepatocyte growth factor according to claim 11, wherein the HEK293 cell is cultured by the following steps: serum-free culture medium is adopted for suspension culture.

13. A host cell transfected or transformed with a plasmid according to any one of claims 1 to 12 expressing a pro-enzyme form of hepatocyte growth factor and/or a plasmid according to any one of claims 1 to 12 expressing an activator of hepatocyte growth factor.