Expression of recombinant human urinary kallidinogenase in CHO cells

Abstract

The invention discloses expression of recombinant human urinary kallidinogenase in CHO (Chinese Hamster Ovary) cells. The signal peptide in the human urinary kallidinogenase with the signal peptide disclosed by the invention is the signal peptide of an Sic / SPI type. The method disclosed by the invention can be used for efficiently expressing the human urinary kallidinogenase.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the expression of recombinant human prourokinase in CHO cells. Background Art

[0002] Since the 21st century, with the rapid development of the national economy and the improvement of the national living standard, the incidence of cardiovascular and cerebrovascular diseases has risen rapidly, bringing a great burden to people's lives. How to actively and effectively treat cardiovascular and cerebrovascular diseases and ischemic brain injury, clarify their action mechanisms, and find effective and efficient therapeutic drugs is one of the scientific problems faced by people.

[0003] In 1909, Abelous et al. first reported that intravenous injection of human urine could cause a transient decrease in the blood pressure of dogs and found that there was a blood pressure-lowering substance in urine. In 1930, Kraut et al. found a high concentration of this substance in the pancreas and named it "Kallikrein", that is, kallikrein (KLK).

[0004] Subsequently, the discovery of the kallikrein-kinin system (KKS) and its participation in regulating important physiological functions of the cardiovascular, renal, nervous systems, etc. have also been elaborated one by one. KKS is one of the main blood pressure-lowering systems in the body and consists of kininogen, kallikrein, kininase and kinin. Among them, kallikrein (KLK), also known as kininogenase, is the main rate-limiting enzyme of the kinin system. It is a group of serine proteases present in most tissues and body fluids and is an endopeptidase. It specifically cleaves the substrate peptide at the carbon terminus, can cleave kininogen to release active kinin, and the kinin plays a regulatory role in the cardiovascular system and renal function.

[0005] KLK is divided into two major categories, plasma KLK and tissue KLK, which are converted from prekallikrein and prokallikrein, respectively. 1) Plasma KLK, also known as Fletcher factor, is specifically expressed in hepatocytes and is a high-molecular-weight glycoprotein that releases a nonapeptide, namely BK (Bradykinin), using high-molecular-weight kininogen (HMWK) as a substrate. BK can regulate vascular tone, inflammatory responses, and the processes of endogenous blood coagulation and fibrinolysis. 2) Tissue KLK is mainly distributed in the lungs, kidneys, blood vessels, brain, and adrenal tissues and is a medium-sized glycoprotein. Among all known tissue kallikrein families, only pancreatic / kidney KLK can release active kinins from kininogens. It mainly uses low-molecular-weight kininogen (LMWK) as a substrate to release a decapeptide, lysyl bradykinin, which is usually called kinin. The in vivo activity of kinin is stronger than that of the nonapeptide (BK), and it can be cleaved by aminopeptidase to BK to continue to play its role. Kinins can participate in a variety of physiological processes and regulate physiological or pathological processes such as blood pressure regulation, electrolyte balance, and inflammatory responses.

[0006] Human tissue kallikrein (HTK) is widely present in human organs such as the kidneys, cardiovascular system, central nervous system, pancreas, and intestines, and exerts its extensive pathophysiological effects by binding to receptors through its metabolites. Among them, the research on HTK in cardiovascular and kidney diseases is the most extensive. Among them, human urinary kallikrein (HUK) is a serine protease composed of 238 amino acids extracted from the fresh urine of healthy adult men. It acts on related receptors by promoting the cleavage of kininogen by the substrate to produce vasodilator kinin. Kinins are present in very small amounts in normal human bodies, but play a very important role in maintaining normal blood pressure and smooth blood circulation in the human body. Vasodilator plays antioxidant, anti-inflammatory, anti-apoptotic, and promotes neurovascular regeneration and other effects during acute ischemia and hypoxia of the brain tissue, thereby playing a neuroprotective role.

[0007] In addition, a large number of literature reports have shown that HUK can promote the generation of new blood vessels after acute stroke. In cases of lower limb ischemia, myocardial ischemia, and renal ischemia, HUK has a good effect on promoting angiogenesis. HUK can also inhibit neuronal apoptosis after acute stroke. After stroke, the necrosis and apoptosis of a large number of nerve cells are the main causes of cranial nerve function deficits. Effectively inhibiting neuronal apoptosis is the key to stroke treatment. In addition, HUK can significantly inhibit glutamate-induced neuronal morphological changes and neuronal apoptosis. Animal experiments have also found that HUK can increase the level of Bcl2 and reduce the levels of Caspase3 and Box through the classical apoptotic pathway of PI3K / AKT / FoxO1, thereby significantly reducing the neuronal apoptosis rate, reducing the infarct area in the experimental animals after stroke, and promoting nerve function recovery. From the existing reports, it is not difficult to find that human urinary kallidinogenase (HUK) has the following effects: anti-inflammatory, antioxidant, inhibiting neuronal apoptosis, promoting angiogenesis and nerve regeneration in the infarcted area of the brain, promoting glial cell migration, promoting neuroblast differentiation, and reducing the infarct area of the brain, etc.

[0008] Given the important physiological effects of HUK, it is a long and arduous task for researchers to transform it into a clinical product and a drug. Currently, only Guangdong Tianpu Biochemical Pharmaceutical Co., Ltd. has extracted natural human urinary kallidinogenase (HUK) from the fresh urine of healthy men and applied it to clinical treatment. Its trade name is Kelikang: Urinary Kallidinogenase for Injection.

[0009] The CHO cell expression system is currently the most important cell system for the expression of glycosylated proteins, and the protein structure conformation expressed by it is almost identical to that of the natural protein. Currently, there are few studies on expressing human urinary kallidinogenase using the CHO cell expression system, and there is no report on the high-efficiency expression of human urinary kallidinogenase in this system. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to improve the efficiency of expressing human urinary kallidinogenase (HUK) in CHO cells, thereby reducing the production cost.

[0011] The present invention screened out 3 signal peptide sequences that can direct the secretion of HUK extracellularly. After transfecting a eukaryotic cell (such as a CHO cell) with a recombinant expression plasmid encoding HUK with one of these 3 signal peptides, through culturing under specific conditions, the supernatant of the cell culture medium was obtained. After detecting the enzyme activity of HUK in it, it was found that both the enzyme activity during the process and the enzyme activity of the final sample collected were much higher than the enzyme activity of the reference product (Kelikang: Urinary Kallidinogenase for Injection), indicating that the expression efficiency of human urinary kallidinogenase (HUK) in the present invention is very high.

[0012] In a first aspect, the present invention provides a human urinary kallikrein with a signal peptide, wherein the signal peptide is a Sec / SPI type signal peptide, which is a "standard" secretion signal peptide transported by the Sec transposon and cleaved by signal peptidase I (Lep).

[0013] As used herein, the term "signal peptide", also called signal sequence, is a short (5 - 30 amino acids in length) peptide chain that guides the newly synthesized protein to the secretory pathway. It is usually located at the amino terminus of the protein and is a localization tag for membrane proteins, secreted proteins, and lysosomal proteins. Before expressing a recombinant protein, it is usually necessary to predict the signal peptide of the protein's amino acid sequence.

[0014] The amino acid sequence of the human urinary kallikrein described herein is shown in SEQ ID NO:1, consisting of 238 amino acids, containing 5 pairs of S - S bonds and 3 glycosylation sites, and its primary structure is as Figure 1 shown.

[0015] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, or a signal peptide having one or more amino acid substitutions, deletions, and / or additions relative to these sequences, or a signal peptide having a sequence identity of more than 80% thereto. As used herein, the term "a plurality of amino acids" refers to, for example, 2 to 4 amino acid residues, preferably 2 to 3 amino acid residues, and most preferably 2 amino acid residues. As used herein, the term "sequence identity of more than 80%" can be more than 80%, preferably more than 85%, more preferably more than 90%. "Sequence identity" means the percentage of identical bases (or amino acids) when comparing two sequences after alignment. Such alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 2007). Preferably, alignment is performed using default parameters. One alignment program is BLAST using default parameters.

[0016] In some embodiments, in the above - mentioned human urinary kallikrein with a signal peptide, the amino acid sequence of the signal peptide is shown in SEQ ID NO:2, the probability of having a signal peptide is 99.925%, the signal peptide type is SP (Sec / SPI); the cleavage site is 24 - 25, with a probability of 90.610%. The human urinary kallikrein with this signal peptide is also called HUK1 in the present invention, and its sequence is shown in SEQ ID NO:3.

[0017] In some embodiments, in the above-mentioned human urinary kallikrein with a signal peptide, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:4, the probability of having a signal peptide is 99.726%, the signal peptide type is SP (Sec / SPI); the cleavage site is 17-18, with a probability of 88.830%. The human urinary kallikrein with this signal peptide is also referred to as HUK2 in the present invention, and its sequence is as shown in SEQ ID NO:5.

[0018] In some embodiments, in the above-mentioned human urinary kallikrein with a signal peptide, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:6, the probability of having a signal peptide is 99.086%, the signal peptide type is SP (Sec / SPI); the cleavage site is 19-20, with a probability of 81.690%. The human urinary kallikrein with this signal peptide is also referred to as HUK3 in the present invention, and its sequence is as shown in SEQ ID NO:7.

[0019] In a second aspect, the present invention provides a nucleic acid molecule encoding any one of the above-mentioned human urinary kallikreins with a signal peptide.

[0020] When applied to polynucleotides, the term "encoding" refers to a nucleic acid molecule that can be transcribed and / or translated to produce a polypeptide and / or a fragment thereof if in its natural state or when manipulated by methods well known to those skilled in the art. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0021] In the present invention, the nucleotide sequence of the Sec / SPI type signal peptide is not limited to the following DNA molecules:

[0022] 1) The DNA molecule shown by positions 1-72 in SEQ ID NO:8, positions 1-51 in SEQ ID NO:9 or positions 1-57 in SEQ ID NO:10;

[0023] 2) A DNA molecule that hybridizes with the DNA molecule defined in 1) under stringent conditions and encodes any one of the above-mentioned signal peptides;

[0024] 3) A DNA molecule that has more than 80% identity with the DNA molecule defined in 1) or 2) and encodes any one of the above-mentioned signal peptides.

[0025] As used herein, the term "stringent conditions" refers to conditions that form specific hybrids but never form non-specific hybrids. For example, such conditions include hybridization at 45°C in 6×SSC (sodium chloride / sodium citrate), followed by washing at 50°C to 65°C in 0.2 to 1×SSC and 0.1% SDS. Optionally, such conditions include hybridization at 65°C to 70°C in 1×SSC, followed by washing at 65°C to 70°C in 0.3×SSC. Hybridization can be carried out by conventionally known methods, such as those described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (1989).

[0026] In some embodiments, in the above nucleic acid molecule, the nucleotide sequence encoding the signal peptide of the SP (Sec / SPI) type is upstream of the nucleotide sequence encoding HUK, and preferably the nucleotide sequence encoding the signal peptide of the SP (Sec / SPI) type is immediately upstream of the 5' end of the nucleotide sequence encoding HUK.

[0027] The signal peptide of the present invention is coupled to the N-terminal side of HUK, allowing it to efficiently express HUK in eukaryotic cells (such as mammalian cells). When the protein is expressed in eukaryotic cells (such as mammalian cells), a fusion protein of HUK and the signal peptide is expressed, and subsequently the signal peptide region is cleaved from it by signal peptidase to form the mature protein HUK.

[0028] In some embodiments, in the above nucleic acid molecule, the signal peptide is the signal peptide shown in SEQ ID NO:2, and the nucleotide sequence encoding this signal peptide is shown as positions 1-72 in SEQ ID NO:8; in some embodiments, the nucleic acid molecule is as shown in SEQ ID NO:8, which encodes the above HUK1, positions 1-72 in SEQ ID NO:8 are the nucleotide sequence encoding the signal peptide shown in SEQ ID NO:2, and positions 73-789 are the nucleotide sequence encoding HUK.

[0029] In some embodiments, in the above nucleic acid molecule, the signal peptide is the signal peptide shown in SEQ ID NO:4, and the nucleotide sequence encoding this signal peptide is shown as positions 1-51 in SEQ ID NO:9; in some embodiments, the nucleic acid molecule is as shown in SEQ ID NO:9, which encodes the above HUK2, positions 1-51 in SEQ ID NO:9 are the nucleotide sequence encoding the signal peptide shown in SEQ ID NO:4, and positions 52-768 are the nucleotide sequence encoding HUK.

[0030] In some embodiments, in the above nucleic acid molecule, the signal peptide is the signal peptide shown in SEQ ID NO: 6, and the nucleotide sequence encoding this signal peptide is as shown at positions 1-57 in SEQ ID NO: 10; in some embodiments, the nucleic acid molecule is as shown in SEQ ID NO: 10, which encodes the above-mentioned HUK3, positions 1-57 in SEQ ID NO: 10 are the nucleotide sequence encoding the signal peptide shown in SEQ ID NO: 6, and positions 58-774 are the nucleotide sequence encoding HUK.

[0031] The nucleic acid molecule provided by the present invention can generally be obtained by PCR amplification or artificial synthesis methods.

[0032] In a third aspect, the present invention provides a recombinant vector, which contains any one of the above-mentioned nucleic acid molecules;

[0033] The recombinant vector includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes.

[0034] In some embodiments, the recombinant vector is a recombinant expression vector, preferably a non-viral vector, more preferably a mammalian cell expression vector, such as pCGS3.2, pEE12.4, pcDNA3.1 series vectors.

[0035] By conventional known techniques, the nucleic acid encoding human urokinase plasminogen activator with a signal peptide is incorporated into an expression vector, and the recombinant expression vector thus obtained is introduced into eukaryotic cells (such as mammalian cells), which enables the accumulation of the protein with a signal peptide in the cells.

[0036] In some embodiments, the recombinant expression vector is obtained by inserting the nucleotide of the recombinant HUK into the multiple cloning site of pcDNA3.1(+), resulting in a recombinant expression plasmid.

[0037] In some embodiments, the recombinant expression vector is pcDNA3.1-HUK1, which is obtained by inserting the DNA molecule shown in SEQ ID NO: 8 into the multiple cloning site of pcDNA3.1(+).

[0038] In some embodiments, the recombinant expression vector is pcDNA3.1-HUK2, which is obtained by inserting the DNA molecule shown in SEQ ID NO: 9 into the multiple cloning site of pcDNA3.1(+).

[0039] In some embodiments, the recombinant expression vector is pcDNA3.1-HUK3, which is obtained by inserting the DNA molecule shown in SEQ ID NO: 10 into the multiple cloning site of pcDNA3.1(+).

[0040] In addition to the nucleic acid encoding human urinary kallikreinogenase with a signal peptide, the recombinant expression vector of the present invention may not only include a promoter that initiates the gene transcription of human urinary kallikreinogenase with a signal peptide, such as the CMV promoter, SV40 promoter; but also include a terminator that terminates the gene transcription of human urinary kallikreinogenase with a signal peptide, and may also include an enhancer sequence, such as the CMV enhancer sequence.

[0041] The method for constructing the recombinant expression vector can be any known method. The above-described promoter, the nucleic acid encoding human urinary kallikreinogenase with a signal peptide, and other DNA segments (such as terminators, enhancers) if present can be introduced into a suitably selected vector used as a basis in a predetermined order. For example, a recombinant vector can be constructed by using restriction endonucleases and ligases, etc.

[0042] In a fourth aspect, the present invention provides a recombinant cell comprising any one of the above-described nucleic acid molecules and / or any one of the above-described recombinant vectors.

[0043] In some embodiments, the recombinant cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell, such as human embryonic kidney cell HEK293, human embryonic retina cell PER.C6, MDCK cell, African green monkey kidney cell COS, mouse myeloma cell NS0 and Sp2 / 0, hamster kidney cell BHK-21, and Chinese hamster ovary cell (CHO cell).

[0044] In some embodiments, the recombinant cell is the CHO cell 1 of the present invention, which is obtained by transferring pcDNA3.1-HUK1 into CHO cells.

[0045] In some embodiments, the recombinant cell is the CHO cell 2 of the present invention, which is obtained by transferring pcDNA3.1-HUK2 into CHO cells.

[0046] In some embodiments, the recombinant cell is the CHO cell 3 of the present invention, which is obtained by transferring pcDNA3.1-HUK3 into CHO cells.

[0047] Currently, the commonly used cell transfection methods are mainly divided into three categories: physical mediation (electroporation method, gene gun method, microinjection method), chemical mediation (liposome transfection method, calcium phosphate co-precipitation method, cationic polymer-mediated method), and biological mediation (virus-mediated transfection, protoplast transfection). The above recombinant expression vector is introduced into target cells by conventional transfection methods.

[0048] In some embodiments, the recombinant expression vector is transiently transfected into CHO cells by electroporation (electroporation method). For example, according to the ratio of electroporation Buffer: plasmid: number of CHO cells = 1 mL: 100 μg: 1×10⁸, the recombinant expression vectors pcDNA3.1-HUK1, pcDNA3.1-HUK2, and pcDNA3.1-HUK3 are respectively transiently transfected into CHO cells (the density of the CHO cell culture medium is 7×10⁶ - 9×10⁶ cells / mL, such as 7.0×10⁶, 7.1×10⁶, 7.2×10⁶, 7.3×10⁶, 7.4×10⁶, 7.5×10⁶, 7.6×10⁶, 7.7×10⁶, 7.8×10⁶, 7.9×10⁶, 8.0×10⁶, 8.1×10⁶, 8.2×10⁶, 8.3×10⁶, 8.4×10⁶, 8.5×10⁶, 8.6×10⁶, 8.7×10⁶, 8.8×10⁶, 8.9×10⁶, 9.0×10⁶ cells / mL, or any value and range between any two of these values), to obtain CHO cell 1 transfected with the recombinant expression plasmid pcDNA3.1-HUK1, CHO cell 2 transfected with the recombinant expression plasmid pcDNA3.1-HUK2, and CHO cell 3 transfected with the recombinant expression plasmid pcDNA3.1-HUK3.

[0049] In a fifth aspect, the present invention provides a method for preparing human urinary kallidinogenase HUK, comprising the following steps:

[0050] Culturing the recombinant cells under conditions that enable the expression of human urinary kallidinogenase in any of the above-mentioned recombinant cells, harvesting the supernatant of the cell culture medium, and obtaining human urinary kallidinogenase.

[0051] In some embodiments, in the above preparation method, it further includes the step of separating and purifying HUK from the supernatant.

[0052] In some embodiments, in any of the above-mentioned preparation methods, the culturing includes the following steps: culturing the constructed recombinant cells conventionally, when the cell density reaches 6×10⁶ - 10×10⁶ cells / mL, lowering the temperature to enable the large-scale expression of human urinary kallidinogenase; regularly detecting the glucose content in the culture medium and maintaining the glucose content above 2 g / L.

[0053] In some embodiments, in any of the above-mentioned preparation methods, the recombinant cells are CHO cells, and the culturing includes the following steps: culturing the constructed CHO cells conventionally, when the cell density reaches 6×10⁶ - 10×10⁶ cells / mL, lowering the temperature to enable the large-scale expression of human urinary kallidinogenase; regularly detecting the glucose content in the culture medium and ensuring that the glucose content is maintained above 2 g / L;

[0054] Preferably, the conventional culture includes: on the day of transfection (e.g., electroporation), i.e., D0, the transfected CHO cells are inoculated into a CHO cell medium (e.g., CHO CD04 medium) at a viable cell density of 4.5E6 - 5.5E6 cells / mL (e.g., 4.5E6, 4.6E6, 4.7E6, 4.8E6, 4.9E6, 5.0E6, 5.1E6, 5.2E6, 5.3E6, 5.4E6, 5.5E6 cells / mL, or any value and range between any two of these values), and cultured in a shaker at 37°C and 8% CO2 (the rotation speed is, for example, 120 rpm);

[0055] Preferably, the temperature reduction is from 37°C to 32°C;

[0056] Preferably, the glucose content is maintained above 2 g / L by adding a feeding medium containing glucose (e.g., CHOFeed 03 feeding, one or both of CHO Feed02 feeding) and / or an aqueous glucose solution (e.g., an aqueous glucose solution with a concentration of 300 g / L) during the culture process; in some embodiments, when glucose supplementation is first required, a feeding medium containing glucose is added, when glucose supplementation is second required, an aqueous glucose solution is added, when glucose supplementation is third required, a feeding medium containing glucose is added, when glucose supplementation is fourth required, an aqueous glucose solution is added, and then an aqueous glucose solution is added each time glucose supplementation is required to maintain the glucose content above 2 g / L; in some embodiments, the feeding medium containing glucose is added by the following method: The addition ratio of CHO Feed 03 feeding and CHO Feed02 feeding is 8:1 - 12:1 (e.g., 8:1, 9:1, 10:1, 11:1, 12:1, or any ratio and range between any two of these ratios); in some embodiments, the glucose content in the culture solution is maintained at 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any value and range between any two of these values.

[0057] In some embodiments, in any of the above-described preparation methods, the recombinant cell is a CHO cell, and the culture includes the following steps: On the day of transfection (e.g., electroporation) (i.e., D0), the transfected CHO cells are inoculated into a CHO cell medium (e.g., CHO CD04 medium) at a viable cell density of 4.5E6 - 5.5E6 cells / mL (e.g., 4.5E6, 4.6E6, 4.7E6, 4.8E6, 4.9E6, 5.0E6, 5.1E6, 5.2E6, 5.3E6, 5.4E6, 5.5E6 cells / mL, or the values and ranges between any two of these values), and cultured in a shaker at 37°C and 8% CO2 (the rotation speed is, for example, 120 rpm). At D1 and D6, 8% - 12% (e.g., 8%, 9%, 10%, 11%, 12%, or the values and ranges between any two of these values) of the CHO Feed 03 supplement and 0.5% - 1.5% (e.g., 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, or the values and ranges between any two of these values) of the CHO Feed02 supplement (to help the damaged cells recover quickly after electroporation and provide the necessary nutrients for maintaining the viability of CHO cells in the later stage of suspension culture, which can significantly increase the density of CHO cells and the protein expression level in the suspension culture system). When the cell density reaches 6E6 - 10E6 cells / mL (e.g., D3), the cells are transferred to a shaker at 32°C and 5% CO2 (the rotation speed is, for example, 120 rpm) (to inhibit the rapid growth of the cells, thereby turning to the rapid expression of human prourokinase); at D5, D7, and D9, the glucose content is detected. If the glucose content is less than 4 g / L, a glucose aqueous solution with a concentration of 300 g / L is added to make up the glucose content to 7 g / L.

[0058] In some embodiments, in any of the above-described preparation methods, after the temperature reduction, the culture is terminated when the cell viability drops to 60% or before the cell viability drops to 60%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 61% or before the cell viability drops to 61%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 62% or before the cell viability drops to 62%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 63% or before the cell viability drops to 63%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 64% or before the cell viability drops to 64%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 65% or before the cell viability drops to 65%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 66% or before the cell viability drops to 66%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 67% or before the cell viability drops to 67%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 68% or before the cell viability drops to 68%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 69% or before the cell viability drops to 69%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 70% or before the cell viability drops to 70%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 71% or before the cell viability drops to 71%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 72% or before the cell viability drops to 72%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 73% or before the cell viability drops to 73%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 74% or before the cell viability drops to 74%; in some embodiments, after the temperature reduction, the culture is terminated when the cell viability drops to 75% or before the cell viability drops to 75%.

[0059] In some embodiments, in any of the above-described preparation methods, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.9E6 cells / mL or before, the culture is terminated; in some embodiments, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.8E6 cells / mL or before, the culture is terminated; in some embodiments, when the cell viability drops to the specified viability and when the cell density drops to 7.7E6 cells / mL or before, the culture is terminated. In some embodiments, when the cell viability drops to the specified viability and when the cell density drops to 7.6E6 cells / mL or before, the culture is terminated; in some embodiments, when the cell viability drops to the specified viability and when the cell density drops to 7.5E6 cells / mL or before, the culture is terminated; in some embodiments, when the cell viability drops to the specified viability and when the cell density drops to 7.4E6 cells / mL or before, the culture is terminated; in some embodiments, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.3E6 cells / mL or before, the culture is terminated; in some embodiments, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.2E6 cells / mL or before, the culture is terminated; in some embodiments, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.1E6 cells / mL or before, the culture is terminated; in some embodiments, after the temperature reduction, when the cell viability drops to the specified viability and when the cell density drops to 7.0E6 cells / mL or before, the culture is terminated.

[0060] In some embodiments, in any of the above-described preparation methods, the number of days of the culture is more than 4 days; in some embodiments, the number of days of the culture is more than 5 days; in some embodiments, the number of days of the culture is more than 6 days; in some embodiments, the number of days of the culture is more than 7 days; in some embodiments, the number of days of the culture is more than 8 days; in some embodiments, the number of days of the culture is more than 9 days; in some embodiments, the number of days of the culture is more than 10 days; in some embodiments, the number of days of the culture is more than 11 days; in some embodiments, the number of days of the culture is more than 12 days; in some embodiments, the number of days of the culture is more than 13 days; in some embodiments, the number of days of the culture is more than 14 days; in some embodiments, the number of days of the culture is more than 15 days.

[0061] In a sixth aspect, the present invention provides the use of a Sec / SPI type signal peptide in the preparation of human urinary kallikrein.

[0062] In some embodiments, in the above application, the signal peptide is the signal peptide shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a signal peptide having one or more amino acid substitutions, deletions and / or additions relative to these sequences, or a signal peptide having a sequence identity of more than 80% thereto.

[0063] In the present invention, the gene of human urinary kallikrein is cloned by molecular biological means, a signal peptide sequence is added, the gene encoding human urinary kallikrein with a signal peptide is transferred into an in vitro mammalian cell for recombinant expression, and then the cell culture technology is used to culture the cells so that the recombinant protein is highly expressed, and human urinary kallikrein is obtained from the extracellular secretion of the cells.

[0064] The method provided by the present invention can highly express HUK. This method not only completely gets rid of the shackles of the limited source of raw materials in the existing process, but also realizes the purpose of highly expressing human urinary kallikrein products in a short time. The present invention greatly reduces the production cost of HUK and makes the necessary technical preparations for the later industrial production of HUK. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 is the active amino acid sequence of urikinase.

[0066] Figure 2 are the top three signal peptides with a probability of recombinant HUK being screened out.

[0067] Figure 3 is the light absorption value of each group during enzyme activity detection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0068] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0069] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0070] The following further illustrates the present invention in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0071] Examples

[0072] 1. Obtaining the amino acid sequence of human urinary kallikrein (HUK)

[0073] The amino acid sequence of human urinary kallikrein (HUK) (Sequence ID: prf||1409287A) is found from NCBI and is shown as SEQ ID NO: 1.

[0074] IVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTAAHCISDNYQLWLGRHNLFD

[0075] DENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDLMLLRLTEPADTITDAVKVVE

[0076] LPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDECEKAHVQKVTDFMLCVG

[0077] HLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS(SEQ IDNO:1)

[0078] This sequence consists of 238 amino acids, contains 5 pairs of S-S bonds and 3 glycosylation sites, and its primary structure is as Figure 1 shown.

[0079] 2. Screening of signal peptides

[0080] Using the NovoPro protein signal peptide prediction tool, through comparison, 3 signal peptide sequences that can direct the secretion of HUK extracellularly were finally screened out. The sequences, types, probabilities and cleavage sites of these 3 signal peptides are as Figure 2 shown. Among them, the probability of having a signal peptide refers to the probability that the protein is a secreted protein and there is a short peptide of signal peptide that can direct its secretion extracellularly.

[0081] The cleavage site of the first signal peptide is between the 24th and 25th amino acids, and the following probability is the possibility of predicting the cleavage site of the signal peptide at this position.

[0082] The cleavage site of the second signal peptide is between the 17th and 18th amino acids, and the following probability is the possibility of predicting the cleavage site of the signal peptide at this position.

[0083] The cleavage site of the third signal peptide is between the 19th and 20th amino acids, and the following probability is the possibility of predicting the cleavage site of the signal peptide at this position.

[0084] The information of the 3 screened signal peptides is as follows:

[0085] (1) The amino acid sequence of the first signal peptide is MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 2), the probability of having a signal peptide is 99.925%, the signal peptide type is SP (Sec / SPI); the cleavage site is 24 - 25, with a probability of 90.610%. The HUK linked to this signal peptide is abbreviated as HUK1, and its sequence is as shown in SEQ ID NO: 3.

[0086] MALWMRLLPLLALLALWGPDPAAAIVGGWECEQHSQPWQAALYHFSTFQCGGILVHR

[0087] QWVLTAAHCISDNYQLWLGRHNLFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADE

[0088] DYSHDLMLLRLTEPADTITDAVKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCV

[0089] DLKILPNDECEKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS(SEQ ID NO:3)

[0090] (2) The amino acid sequence of the second signal peptide is MRAWIFFLLCLAGRALA (SEQ ID NO: 4), the probability of having a signal peptide is 99.726%, the signal peptide type is SP (Sec / SPI); the cleavage site is 17 - 18, with a probability of 88.830%. The HUK linked to this signal peptide is abbreviated as HUK2, and its sequence is as shown in SEQ ID NO: 5.

[0091] MRAWIFFLLCLAGRALAIVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTAAH

[0092] CISDNYQLWLGRHNLFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDLML

[0093] LRLTEPADTITDAVKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPNDE

[0094] CEKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS(SEQ ID NO:5)

[0095] (3) The amino acid sequence of the third signal peptide is MGWSCIILFLVATATGVHS (SEQ ID NO:6), the probability of having a signal peptide is: 99.086%, signal peptide type: SP (Sec / SPI); cleavage site: 19 - 20, probability 81.690%. Abbreviate the HUK connected with this signal peptide as HUK3, and its sequence is as shown in SEQ ID NO:7.

[0096] MGWSCIILFLVATATGVHSIVGGWECEQHSQPWQAALYHFSTFQCGGILVHRQWVLTA

[0097] AHCISDNYQLWLGRHNLFDDENTAQFVHVSESFPHPGFNMSLLENHTRQADEDYSHDL

[0098] MLLRLTEPADTITDAVKVVELPTQEPEVGSTCLASGWGSIEPENFSFPDDLQCVDLKILPN

[0099] DECEKAHVQKVTDFMLCVGHLEGGKDTCVGDSGGPLMCDGVLQGVTSWGYVPCGTPNKPSVAVRVLSYVKWIEDTIAENS(SEQ ID NO:7)

[0100] 3. Construction of recombinant expression plasmid

[0101] (1) Synthesize the coding gene sequence of HUK1, as shown in SEQ ID NO:8.

[0102] atggcgctgtggatgcgcctgctgccgctgctggcgctgctggcgctgtggggcccggatccggcggcggcgattgtgggcggctggga

[0103] atgcgaacagcatagccagccgtggcaggcggcgctgtatcattttagcacctttcagtgcggcggcattctggtgcatcgccagtgggtgc

[0104] tgaccgcggcgcattgcattagcgataactatcagctgtggctgggccgccataacctgtttgatgatgaaaacaccgcgcagtttgtgcatgt

[0105] gagcgaaagctttccgcatccgggctttaacatgagcctgctggaaaaccatacccgccaggcggatgaagattatagccatgatctgatgc

[0106] tgctgcgcctgaccgaaccggcggataccattaccgatgcggtgaaagtggtggaactgccgacccaggaaccggaagtgggcagcacc

[0107] tgcctggcgagcggctggggcagcattgaaccggaaaactttagctttccggatgatctgcagtgcgtggatctgaaaattctgccgaacga

[0108] tgaatgcgaaaaagcgcatgtgcagaaagtgaccgattttatgctgtgcgtgggccatctggaaggcggcaaagatacctgcgtgggcgat

[0109] agcggcggcccgctgatgtgcgatggcgtgctgcagggcgtgaccagctggggctatgtgccgtgcggcaccccgaacaaaccgagcgtggcggtgcgcgtgctgagctatgtgaaatggattgaagataccattgcggaaaacagctaa(SEQ ID NO:8)

[0110] The nucleotide sequence encoding the first signal peptide is located at positions 1 - 72 of SEQ ID NO:8, and the nucleotide sequence encoding HUK is located at positions 73 - 789 of SEQ ID NO:8.

[0111] The DNA molecule shown in SEQ ID NO:8 was inserted into the multiple cloning site of pcDNA3.1(+) to obtain the recombinant expression plasmid pcDNA3.1 - HUK1.

[0112] (2) Synthesize the coding gene sequence of HUK2, as shown in SEQ ID NO:9.

[0113] atgcgcgcgtggattttttttctgctgtgcctggcgggccgcgcgctggcgattgtgggcggctgggaatgcgaacagcatagccagccgtg

[0114] gcaggcggcgctgtatcattttagcacctttcagtgcggcggcattctggtgcatcgccagtgggtgctgaccgcggcgcattgcattagcga

[0115] taactatcagctgtggctgggccgccataacctgtttgatgatgaaaacaccgcgcagtttgtgcatgtgagcgaaagctttccgcatccggg

[0116] ctttaacatgagcctgctggaaaaccatacccgccaggcggatgaagattatagccatgatctgatgctgctgcgcctgaccgaaccggcg

[0117] gataccattaccgatgcggtgaaagtggtggaactgccgacccaggaaccggaagtgggcagcacctgcctggcgagcggctggggca

[0118] gcattgaaccggaaaactttagctttccggatgatctgcagtgcgtggatctgaaaattctgccgaacgatgaatgcgaaaaagcgcatgtgc

[0119] agaaagtgaccgattttatgctgtgcgtgggccatctggaaggcggcaaagatacctgcgtgggcgatagcggcggcccgctgatgtgcg

[0120] atggcgtgctgcagggcgtgaccagctggggctatgtgccgtgcggcaccccgaacaaaccgagcgtggcggtgcgcgtgctgagctatgtgaaatggattgaagataccattgcggaaaacagctaa(SEQ ID NO:9)

[0121] The nucleotide sequence encoding the second signal peptide is located at positions 1 - 51 of SEQ ID NO:9, and the nucleotide sequence encoding HUK is located at positions 52 - 768 of SEQ ID NO:9.

[0122] The DNA molecule shown in SEQ ID NO:9 was inserted into the multiple cloning site of pcDNA3.1(+), resulting in the recombinant expression plasmid pcDNA3.1 - HUK2.

[0123] (3) Synthesize the coding gene sequence of HUK3, as shown in SEQ ID NO:10.

[0124] atgggctggagctgcattattctgtttctggtggcgaccgcgaccggcgtgcatagcattgtgggcggctgggaatgcgaacagcatagcca

[0125] gccgtggcaggcggcgctgtatcattttagcacctttcagtgcggcggcattctggtgcatcgccagtgggtgctgaccgcggcgcattgca

[0126] ttagcgataactatcagctgtggctgggccgccataacctgtttgatgatgaaaacaccgcgcagtttgtgcatgtgagcgaaagctttccgca

[0127] tccgggctttaacatgagcctgctggaaaaccatacccgccaggcggatgaagattatagccatgatctgatgctgctgcgcctgaccgaac

[0128] cggcggataccattaccgatgcggtgaaagtggtggaactgccgacccaggaaccggaagtgggcagcacctgcctggcgagcggctg

[0129] gggcagcattgaaccggaaaactttagctttccggatgatctgcagtgcgtggatctgaaaattctgccgaacgatgaatgcgaaaaagcgc

[0130] atgtgcagaaagtgaccgattttatgctgtgcgtgggccatctggaaggcggcaaagatacctgcgtgggcgatagcggcggcccgctgat

[0131] gtgcgatggcgtgctgcagggcgtgaccagctggggctatgtgccgtgcggcaccccgaacaaaccgagcgtggcggtgcgcgtgctgagctatgtgaaatggattgaagataccattgcggaaaacagctaa(SEQ ID NO:10)

[0132] Positions 1 - 57 of SEQ ID NO:10 are the nucleotide sequence encoding the third signal peptide, and positions 58 - 774 are the nucleotide sequence encoding HUK.

[0133] The DNA molecule shown in SEQ ID NO:10 was inserted into the multiple cloning site of pcDNA3.1(+) to obtain the recombinant expression plasmid pcDNA3.1 - HUK3.

[0134] The recombinant expression plasmid was sequenced and verified, and the sequence was correct and consistent with the expectation.

[0135] The recombinant expression plasmids pcDNA3.1 - HUK1, pcDNA3.1 - HUK2, and pcDNA3.1 - HUK3 were filter - sterilized.

[0136] 4. Construction and culture of CHO cells expressing recombinant HUK

[0137] After resuscitating the CHO cells, they were passaged more than 3 generations before transfection:

[0138] (1) On the day of transfection D0, according to the ratio of electroporation buffer: plasmid: number of CHO cells = 1 mL: 100 μg: 1×10⁸, 1.5 mg of the recombinant expression plasmids pcDNA3.1 - HUK1, pcDNA3.1 - HUK2, and pcDNA3.1 - HUK3 were transiently transfected into CHO cells (the density of CHO cell culture medium was 8×10⁶ cells / mL) by electroporation, respectively, to obtain CHO cell 1 transfected with the recombinant expression plasmid pcDNA3.1 - HUK1, CHO cell 2 transfected with the recombinant expression plasmid pcDNA3.1 - HUK2, and CHO cell 3 transfected with the recombinant expression plasmid pcDNA3.1 - HUK3.

[0139] (2) After cell electroporation, the cells need to be placed in a cell incubator at 37°C and 8% CO2 for static incubation for 20 min, sampled, and then counted using a Vi-CELL automatic cell counter. The cells are inoculated into CHO CD04 medium (Zhongshan Kangtianshenghe Biotechnology Co., Ltd., product number: A11004), and then cultured in a cell culture flask on a shaker at 37°C, 8% CO2, and 120 rpm.

[0140] (3) On D1, within 24 h after electroporation, add 10% of the cell culture medium volume of CHO Feed03 supplement (Zhongshan Kangtianshenghe Biotechnology Co., Ltd., product number: A11903) and 1% of CHO Feed02 supplement (Zhongshan Kangtianshenghe Biotechnology Co., Ltd., product number: A11902).

[0141] (4) On D3, when the cell density reaches 6E6 - 7E6 cells / mL, transfer the cell sample to a shaker at 32°C, 5% CO2, and 120 rpm for culture, and all subsequent cultures are carried out under this culture condition.

[0142] (5) Subsequently, on D6, add 10% of the cell culture medium volume of CHO Feed 03 supplement and 1% of CHO Feed02 supplement again in the same way as on D1.

[0143] (6) During the culture period, on D5, D7, and D9, use a Roche Cedex Bio biochemical analyzer to detect the glucose content in the cell culture medium. If the glucose content is less than 4 g / L, add a glucose aqueous solution (concentration: 300 g / L) to make up the glucose content to 7 g / L to maintain normal cell growth and protein expression. During the entire culture period, use a Vi-CELL automatic cell counter to monitor the cell density and viability at any time.

[0144] (7) On D11, when the cell density is 7E6 - 8E6 cells / mL and the viability drops below 70%, collect the samples at this time. Take out the cell suspension and place it in a centrifuge tube. Then centrifuge at 4°C and 5000 rpm for 20 min, take out the supernatant and transfer it to a new collector. It can be temporarily frozen at -80°C when not for detection.

[0145] The cell density and viability during the cell culture process after transient transfection are shown in Table 1.

[0146] Table 1 Cell density and viability of CHO cells transfected with recombinant expression plasmids pcDNA3.1-HUK1, pcDNA3.1-HUK2, and pcDNA3.1-HUK3

[0147]

[0148]

[0149] After the electroporation, the seeding density of CHO cells was 5E6 cells / mL. As the number of culture days increased, the density of CHO cells gradually increased and reached its peak on D6-D7. Subsequently, the cell density decreased slightly, and the cell viability showed a gradually decreasing trend from after electroporation until sample collection. On D10, the viability decreased to nearly 70%, and on D11, the viability dropped below 70%.

[0150] 5. Enzyme activity detection of recombinant human prourokinase

[0151] Perform enzyme activity detection of recombinant human prourokinase on the process sample supernatants of CHO cell 1, CHO cell 2, and CHO cell 3 at D5 (i.e., HUK1-D5, HUK2-D5, HUK3-D5), the process sample supernatants of CHO cell 1, CHO cell 2, and CHO cell 3 at D7 (i.e., HUK1-D7, HUK2-D7, HUK3-D7), the process sample supernatants of CHO cell 1, CHO cell 2, and CHO cell 3 at D9 (i.e., HUK1-D9, HUK2-D9, HUK3-D9), and the final sample supernatants of CHO cell 1, CHO cell 2, and CHO cell 3 at D11 (i.e., HUK1-D11, HUK2-D11, HUK3-D11). The specific steps are as follows:

[0152] (1) Prepare the chromogenic substrate (S-2266 (H-D-Val-Leu-Arg-pNA·2HCl) (Shanghai Boatman Biotechnology Co., Ltd., product number: B2266) in advance. S-2266 is a chromogenic substrate for glandular kallikrein. It is a chemically synthesized small peptide with a chromogenic group p-nitroaniline (pNA) at one end. Human prourokinase can catalyze the dissociation of pNA, and the free pNA can be detected by a spectrophotometer or a microplate reader to determine the activity of human prourokinase.

[0153] (2) Take a 5 mL EP tube and set up a blank control group (Blank), three positive control groups (Reference Standard 1: 5 PNA U, Reference Standard 2: 10 PNA U, Reference Standard 3: 15 PNA U), and groups HUK1-D5, HUK2-D5, HUK3-D5, HUK1-D7, HUK2-D7, HUK3-D7, HUK1-D9, HUK2-D9, HUK3-D9, HUK1-D11, HUK2-D11, and HUK3-D11, with three parallels in each group. Blank control group (Blank): Add 0.2 mL of sodium chloride injection (normal saline); Three positive control groups: Add 0.2 mL of the concentrated solution of the reference standard (Kailikang: Urokinase Proenzyme for Injection) with different enzyme activity units (5, 10, and 15 PNA U respectively) (the solvent is normal saline); Groups HUK1-D5, HUK2-D5, HUK3-D5, HUK1-D7, HUK2-D7, HUK3-D7, HUK1-D9, HUK2-D9, HUK3-D9, HUK1-D11, HUK2-D11, and HUK3-D11: Add 0.2 mL of the corresponding supernatant respectively; Then add 4 mL of Tris-HCl buffer solution with a concentration of 0.2 mol / L (pH 8.0) to each tube, mix well, and incubate in a water bath at 37 °C for 5 min; After the incubation, add 0.4 mL of acetic acid aqueous solution with a volume percentage of 50% to the blank control group, and add 0.4 mL of substrate solution S-2266 to the remaining groups, mix well, and react in a water bath at 37 °C (±0.5 °C) for 15 min; After the water bath, add 0.4 mL of substrate solution S-2266 to the blank control group, and add 0.4 mL of acetic acid aqueous solution with a volume percentage of 50% to the remaining groups to terminate the reaction; Finally, use an enzyme-labeled instrument to measure the light absorption value at a wavelength of 405 nm. Using the light absorption value of the blank control group as the blank, measure the absorption value A, and control the A value of the blank control group between 0.1 and 0.2.

[0154] The light absorption value results of each group are as Figure 3 shown.

[0155] Substitute the measured light absorption value A of the samples in the control group and the experimental group into the following formula to calculate the enzyme activity: PNA U / mL = 173.6 * A * T / 1000 (T is the dilution factor).

[0156] 1 PNA U: The amount of human urinary kallikrein enzyme that hydrolyzes 1 mol of pNA per minute at 37 °C and pH 8.0 with S-2266 as the substrate.

[0157] The enzyme activity detection results are shown in Table 2.

[0158] Table 2 Enzyme Activity Detection Results

[0159]

[0160] The above enzyme activity results indicate that whether it is the enzyme activity of the process samples of D5, D7, and D9 or the enzyme activity of the final sampling sample of D11, they are all much higher than the enzyme activity of the reference product, which means that the amount of HUK expressed in the CHO culture supernatant is much higher than 15 PNA U of the reference product. Therefore, the recombinant human prourokinase CHO expression system of the present invention can highly express human prourokinase.

Claims

1. A human urinary kallikreinogenase with a signal peptide, wherein the signal peptide is a Sec / SPI type signal peptide.

2. The human urinary kallikrein with a signal peptide according to claim 1, wherein: The amino acid sequence of the signal peptide is as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a signal peptide having one or more amino acid substitutions, deletions and / or additions relative to these sequences, or a signal peptide having a sequence identity of more than 80% thereto; Preferably, the amino acid sequence of the human urinary kallikreinogenase with a signal peptide is as shown in SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:

7.

3. A nucleic acid molecule encoding the human urinary kallikreinogenase with a signal peptide according to claim 1 or 2.

4. A recombinant vector comprising the nucleic acid molecule according to claim 3.

5. A recombinant cell comprising the nucleic acid molecule according to claim 3 and / or the recombinant vector according to claim 4.

6. The recombinant cell according to claim 5, characterized in that: The recombinant cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell, such as human embryonic kidney cell HEK293, human embryonic retina cell PER.C6, MDCK cell, African green monkey kidney cell COS, mouse myeloma cell NS0 and Sp2 / 0, hamster kidney cell BHK-21, Chinese hamster ovary cell (CHO cell).

7. A method for preparing human urinary kallikreinogenase, comprising the following steps: Culturing the recombinant cell under conditions that enable the recombinant cell according to claim 5 or 6 to express human urinary kallikreinogenase, harvesting the supernatant of the cell culture solution, and obtaining human urinary kallikreinogenase.

8. The method according to claim 7, characterized in that: The culturing comprises the following steps: routinely culturing the constructed recombinant cell, when the cell density reaches 6E6 - 10E6 cells / mL, lowering the temperature to enable a large amount of expression of human urinary kallikreinogenase; regularly detecting the glucose content in the culture solution and maintaining the glucose content above 2 g / L.

9. The method according to claim 7 or 8, characterized in that: After the temperature is lowered, the culture is terminated when the cell viability drops to 60% or before the cell viability drops to 60%.

10. Use of a Sec / SPI type signal peptide in the preparation of human urinary kallikreinogenase.

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