CN109504709B

CN109504709B - Albumin expression vector driven by albumin promoter

Info

Publication number: CN109504709B
Application number: CN201811433912.2A
Authority: CN
Inventors: 朱颂成; 倪华; 赵晓梅
Original assignee: Shanghai Anmin Biotechnology Co ltd
Current assignee: Shanghai Anmin Biotechnology Co ltd
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2020-07-24
Anticipated expiration: 2038-11-28
Also published as: CN109504709A

Abstract

The invention discloses a carrier driven by a human serum albumin promoter and used for expressing an albumin gene in cells, and successfully and efficiently expressing exogenous albumin in the cells, wherein the expression carrier consists of a framework and an expression frame loaded on the framework, the expression frame consists of a human serum albumin promoter and a first intron thereof, an albumin gene, an inner ribosome entry site sequence (IRES) and a marker gene, the human serum albumin promoter and the IRES sequence in the carrier drive the albumin gene and the marker gene to be simultaneously expressed, the carrier is used for successfully and efficiently expressing recombinant human serum albumin in human cells, the albumin secreted into a culture solution reaches 750 mg/L, and the purity is over 90 percent.

Description

Albumin expression vector driven by albumin promoter

Technical Field

The invention belongs to the technical field of genetic engineering, discloses an expression vector driven by an albumin promoter, particularly discloses an expression vector driven by a human serum albumin promoter for efficiently expressing recombinant human serum albumin in human cells, and more particularly discloses a bicistronic vector for simultaneously expressing an exogenous human serum albumin gene and a screening marker driven by the human serum albumin promoter and application thereof in efficiently expressing the recombinant human serum albumin in the human cells.

Technical Field

Human Serum Albumin (HSA) is the major, most abundant soluble protein in Human blood and accounts for approximately half of the total Serum protein [1 ]. The main physiological functions of human serum albumin include: 1, increasing blood volume and maintaining plasma oncotic pressure; regulating the dynamic equilibrium of water between the tissue and the blood vessel; 2, transportation and detoxification functions: albumin can bind anions and cations, can transport different substances including fatty acids, cholesterol, thyroid hormone and the like, and can also transport toxic substances to detoxification organs; 3, nutrition supply: tissue proteins and plasma proteins can be interconverted, and albumin can be used as a nitrogen source to provide nutrients to tissues in the event of nitrogen metabolism disorder. Human serum albumin has been used in medical clinics for over 70 years. The method can be mainly used for: 1, shock caused by blood loss wound and burn; 2, elevated cranial pressure caused by cerebral edema and injury; edema or ascites due to cirrhosis and nephropathy; 4, prevention and treatment of hypoproteinemia; 5, hyperbilirubinemia of the newborn; 6, the medicine is used for cardiopulmonary bypass, auxiliary treatment of burn, auxiliary treatment of hemodialysis, adult respiratory distress syndrome and the like.

Medicinal HSA is derived from human blood extraction [2 ]. However, in China, due to the limitation of the pulp yield, HSA is in short supply and demand, and the price is continuously increased. The preparation of HSA by using blood source also has the problems of potential diseases or virus pollution of blood source, large batch-to-batch difference and the like. Therefore, the strategy for producing HSA by genetic engineering is a focus and trend of attention in the industry. The production of HSA from yeast and rice by recombinant DNA techniques has been highly successful [3], and some products have been approved by clinical trials from governmental drug administration [4 ]. However, the use of recombinant HSA as a replacement for blood-derived albumin as a clinical drug still faces a great challenge. Since human serum albumin is an important functional molecule in the human body, theoretically, recombinant human serum albumin produced from human cells would be the best, most likely successful, alternative to blood-derived albumin for clinical use. However, no efficient expression vector for expression in human cells is currently available.

Human serum albumin is produced mainly by the liver and is also expressed somewhat in the kidney [1,2 ]. The human serum albumin gene shows a gradual rise in the development process and shows a characteristic of very stable expression in the liver after the adult. Under specific physiological and pathological conditions, albumin expression can be changed in response to environmental changes such as nutrition and stress of cells. In the regulatory region upstream of the albumin gene, there are a promoter and a number of cis-regulatory elements, both positive and negative, which interact with trans-acting factors such as various signaling molecules in the cell, and thus regulate the expression of albumin [5,6 ].

In mice, cis-elements including promoters, enhancers, distal enhancers, etc. upstream of the albumin gene have been identified in detail. These elements include promoter and enhancer elements near the first intron, an enhancer 1.7kb upstream of the transcription start site, and a distal enhancer 6-7kb upstream. Upstream to the further distal of the gene, there are some elements that regulate negatively [7,8 ]. The albumin gene promoter and its adjacent binding sites containing TATA box, liver nuclear factor 1 (HNF 1) and nuclear factor Y (NF-Y) constitute the core region for promoting albumin gene expression, and this region mainly activates albumin gene expression [7 ]. Due to the stable, durable and high level expression of the albumin gene in hepatocytes. Therefore, this region containing the albumin promoter may be an ideal promoter for stable, efficient and durable expression of foreign genes in human cells [7,9 ]. In the present invention, the applicants have constructed an albumin promoter-driven expression vector for high level expression of human serum albumin in mammalian cells, particularly human cells.

Disclosure of Invention

An object of the present invention is to provide an albumin promoter-driven expression vector, by which human serum albumin is expressed.

It is still another object of the present invention to provide an expression cell which expresses human serum albumin.

It is yet another object of the present invention to provide a method for producing human serum albumin.

It is a further object of the present invention to provide the use of the human serum albumin promoter.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in one aspect, the present invention provides an albumin promoter driven expression vector characterized in that: the expression vector comprises an expression frame, and the expression frame sequentially comprises an albumin promoter and a first intron thereof, an albumin gene, an inner ribosome entry site sequence IRES and a second gene.

As used herein, the following terms and phrases shall have the meanings as set forth below. 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.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "comprising" is used in an inclusive open sense, meaning that other elements, components, may be included.

In the present invention, an "expression cassette" means a combination of a promoter element and other transcription and translation regulatory control elements operably linked thereto. Heterologous gene sequences may be inserted into the expression cassette for the purpose of expressing the gene sequence. The expression cassette is capable of directing transcription, which results in the production of mRNA for the desired gene product. The expression cassette is inserted into a vector to produce an expression vector that directs the expression of the heterologous protein in a host cell.

A "promoter" consists of two basic parts (a core promoter and a proximal promoter). Promoters are located upstream of the coding sequence of a given gene. The core promoter is defined as the smallest nucleotide sequence capable of directing the precise initiation of transcription of a given gene. The core promoter in eukaryotes is responsible for directing initiation by the RNA polymerase II complex. The core promoter is generally depicted as a sequence spanning the transcription initiation site (INR), more specifically a sequence of 5 to 45 nucleotides upstream and downstream of the INR (70-90 nucleotides in total).

In the present invention, an "albumin promoter", preferably a human serum albumin promoter, is used. Human serum albumin is produced mainly by the liver and is also expressed somewhat in the kidney [1,2 ]. The human serum albumin gene shows a gradual rise in the development process and shows a characteristic of very stable expression in the liver after the adult. Under specific physiological and pathological conditions, albumin expression can be changed in response to environmental changes such as nutrition and stress of cells. In the regulatory region upstream of the albumin gene, there are a promoter and a number of cis-regulatory elements, both positive and negative, which interact with trans-acting factors such as various signaling molecules in the cell, and thus regulate the expression of albumin [5,6 ].

In mice, cis-elements including promoters, enhancers, distal enhancers, etc. upstream of the albumin gene have been identified in detail. These elements include promoter and enhancer elements near the first intron, an enhancer 1.7kb upstream of the transcription start site, and a distal enhancer 6-7kb upstream. Upstream to the further distal of the gene, there are some elements that regulate negatively [7,8 ]. The albumin gene promoter and its adjacent binding sites containing TATA box, liver nuclear factor 1 (HNF 1) and nuclear factor Y (NF-Y) constitute the core region for promoting albumin gene expression, and this region mainly activates albumin gene expression [7 ]. Due to the stable, durable and high level expression of the albumin gene in hepatocytes. Therefore, this region containing the albumin promoter may be an ideal promoter for stable, efficient and durable expression of foreign genes in human cells [7,9 ].

In addition, other serum albumin promoters besides the human serum albumin promoter may be used in the concepts of the present invention without departing from the scope of the present invention.

In the present invention, the human serum albumin promoter and its first intron have the nucleotide sequence shown in SEQ ID NO.1, and variants thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 1. The difference is caused by substitution, deletion or addition of a base.

"albumin" is a protein naturally occurring in mammalian plasma, of which it is the most abundant protein. It plays an important role in maintaining the desired osmotic pressure of the blood and, in addition, in the transport of various substances in the bloodstream. Albumins from many species have been characterized, and they share a high degree of sequence and structural homology. "Serum albumin, is synthesized in the liver and is the most abundant protein in vertebrate plasma. The amino acid sequences and the spatial structures of the serum albumins from different sources are very conservative, and the serum albumins have the properties of combining and transporting endogenous and exogenous substances, maintain the blood colloid osmotic pressure, clear free radicals, inhibit platelet aggregation, anticoagulation and other physiological functions.

In the present invention, the albumin may be selected from human albumin, non-human primate albumin (such as chimpanzee albumin, gorilla albumin or macaque albumin, rodent albumin (such as hamster albumin, guinea pig albumin, mouse albumin and rat albumin), bovine albumin (e.g. cow albumin), equine albumin such as horse albumin or donkey albumin, canine albumin, feline albumin, rabbit albumin, goat albumin, sheep albumin, chicken albumin and pig albumin.

Preferably, the albumin of the invention is human serum albumin. It is to be understood, however, that the present invention is not limited to human serum albumin, and that albumin from other animals may be suitable for use in the present invention and are within the scope of the present invention.

The human serum albumin is a single-chain protein composed of 585 amino acids, the molecular weight is 67kDa, the mature human serum albumin is a heart-shaped molecule, and is composed of 3 α -helical domains with similar structures, the concentration of the mature human serum albumin in plasma is about 42 g/L, and the mature human serum albumin accounts for about 60% of the total protein of the plasma.

Preferably, the human serum albumin gene of the invention has a nucleotide sequence shown in SEQ ID NO.2 or a variant sequence thereof, and the human serum albumin gene of the invention has an amino acid sequence shown in SEQ ID NO.6 or a variant sequence thereof.

The term "variant" refers to a gene or polypeptide comprising an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (several) positions in a parent albumin gene or albumin. Substitution means that a different base or amino acid is substituted for the base or amino acid that originally occupied the position; deletion refers to the removal of the base or amino acid that originally occupied the position; and insertion refers to the addition of new bases or amino acids.

In the present invention, it is preferred that the variant human serum albumin gene has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 2. The difference is caused by substitution, deletion or addition of a base.

In the present invention, preferably, the variant human serum albumin has an amino acid sequence with at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 6. The difference is caused by substitution, deletion or addition of amino acids.

In one embodiment of the present invention, the expression vector of the present invention comprises an albumin gene and a marker gene, constituting a dicistronic cistron.

The present invention is not limited to bicistrons, and in one embodiment of the present invention, the expression vector of the present invention comprises one or more albumin genes and/or marker genes, constituting a tricistron or polycistron.

An "Internal Ribosome Entry Site (IRES), which is the nucleic acid sequence originally discovered in 1988 from the RNA genomes of Poliovirus (PV) and encephalomyocarditis virus (EMCV), is generally located in the 5' untranslated region (UTR) of the RNA virus genome. The IRES can allow the mRNA translation machinery to initiate translation from an IRES position in the middle of the mRNA, such that translation of viral proteins can be independent of the 5' cap structure. After IRES sequences are found from viral RNA, IRES are also found in cellular mRNA, and these IRES are found mainly in mRNA of genes involved in cellular responsiveness to stress and other survival processes [10 ].

The presence of the IRES sequence makes it possible to initiate translation of the protein independent of the cap structure at the 5' end of the messenger RNA (mRNA), making it possible to initiate translation directly from the middle of the mRNA. The IRES sequence thus provides a strategy for expressing the products of several genes independently from one mRNA at the same time, rather than by fusion. Based on this feature, it is possible to design a polycistronic expression vector containing several genes with IRES between them, such as bicistronic mRNA (bicistronic mRNA) where the first gene is dependent on the 5' cap structure and the second gene is dependent on IRES to initiate translation, thereby synthesizing two proteins from one mRNA [10,11 ]. In general, the expression of two proteins before and after IRES is proportional, so that the expression of one reporter gene can reflect the expression of the other gene [12 ]. Polycistronic vectors containing three genes in tandem with two IRES sequences can also be designed, the first gene being translated by the cap structure at messenger RNA 5' and the second gene being translated by the IRES.

This approach is very important in many situations. IRES-based viral and non-viral vectors have been successfully used for preclinical and clinical multigene combination therapy, and are powerful tools commonly used for gene manipulation in basic research [13,14 ]. The usual approach is that the gene of interest relies on the 5' cap structure to initiate translation, while the selection marker relies on the IRES to initiate translation. The expression strategy of the vector based on the IRES is obviously superior to the strategy of expressing two genes by respectively adopting different promoters. When two promoters drive the expression of the target gene and the selection gene respectively, one of the two promoters may be selectively expressed or one of the two promoters may be highly expressed while the other is not expressed or is low in expression due to competition between the two promoters. The IRES vector enables expression of the target gene and the selection marker gene at a relatively stable strength.

Large scale production of therapeutic recombinant protein drugs, such as recombinant monoclonal antibodies or enzymes, is commonly achieved in Chinese hamster ovary Cells (CHO). Cell lines stably expressing a target protein were established by introducing an expression cassette containing the target gene and a selection marker gene into the CHO cell genome and selecting cells into which the expression cassette containing the target gene and the selection marker gene was stably inserted from the cell population [15,16 ]. Obtaining cell lines expressing a target protein stably and at high levels from a large population of cells not containing an expression cassette for the target gene depends on selection of a selection marker gene and an efficient selection method. Industrially, dihydrofolate reductase (dhfr) -based Methotrexate (MTX) selection or glutamine synthetase-based MSX selection [17] (GS-based Methionine Sulfoximine (MSX) selection) are two major screening systems currently in use. The two methods have respective advantages and disadvantages, and relatively speaking, the GS-MSX screening system is more advanced. The DHFR screening system is widely used because it can achieve high expression levels by gene amplification. However, it has the disadvantage of requiring multiple rounds of MTX screening at different concentrations and is therefore time consuming [18 ]. The GS screening system only needs one round of MSX screening, thereby saving time. GS-dependent systems can be screened in GS-knockout CHO without MSX, but also in cells without GS knockout using MSX [19 ]. In addition, GS exerts a positive influence on cell growth because it converts ammonia produced by the metabolism of amino acids into glutamine again, thereby eliminating the accumulation of ammonia in the culture broth and eliminating the pH change and toxicity associated with ammonia accumulation [20 ]. Thus, the glutamine synthetase screening system is a more advantageous screening system [21-23 ].

In the present invention, the IRES sequence has a nucleotide sequence shown in SEQ ID NO.3, and variants thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 3. The difference is caused by substitution, deletion or addition of amino acids.

In one embodiment, the expression vector of the invention comprises one IRES. In another embodiment, the invention comprises two or more IRES, thereby constituting a tricistronic or polycistronic molecule.

The "marker gene" is originally a proper term of genetic engineering, but now it has become a basic experimental tool and is widely used in the research of molecular biology, cell biology, developmental biology, etc. The marker gene is a gene of known function or known sequence, and can function as a specific marker, and whether or not the marker gene is expressed can be determined based on its activity. In the genetic engineering sense, it is an important marker for recombinant DNA vectors, usually used to test whether transformation was successful or not; in the sense of gene localization, it is a tool for marking a gene of interest, and is generally used to detect the localization of a gene of interest in a cell. The activity may be detected directly by detecting the activity of the marker protein itself, or indirectly by detecting a metabolite such as a dye that is generated by the activity of the marker protein. The detection may be any of chemical detection (including detection of enzymatic reactions), physical detection (including detection of behavioral analysis), or sensory detection (including detection using visual, tactile, olfactory, auditory, or taste) of the detector.

In the present invention, the type of marker gene or marker protein is not particularly limited as long as the activity can be detected by a method known in the art. Preferably, the host is a marker protein which has a marker for determining the transformant and is less invasive to the transformant. For example, the protein may be a drug-resistant protein, a fluorescent protein, a pigment-synthesizing protein, a luminescent protein, an external secretory protein, a protein for controlling external morphology, or the like. The use of drug resistance genes and fluorescence marker genes is a common and effective screening method.

In some embodiments of the invention, the marker gene is a drug resistance gene, preferably the drug marker gene is selected from the group consisting of human glutamine synthetase gene, kanamycin resistance gene (nptII), tetracycline resistance gene (tetR), neomycin phosphotransferase gene (npt), puromycin resistance gene, preferably human glutamine synthetase gene.

In the present invention, the human glutamine synthetase gene has a nucleotide sequence shown in SEQ ID NO.4 and variants thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 4. The difference is caused by substitution, deletion or addition of a base.

In the present invention, human glutamine synthetase has the amino acid sequence shown in SEQ ID NO.7 and variants thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 7. The difference is caused by substitution, deletion or addition of amino acids.

It is to be understood that the present invention is not limited to the human glutamine synthetase gene, and those skilled in the art can select other suitable drug resistance genes as needed and are within the scope of the present invention.

In some embodiments of the invention, the marker gene is a fluorescent marker gene, preferably, a green fluorescent protein or a red fluorescent protein.

Green fluorescent protein or Red fluorescent protein are important and widely used as visual screening markers for monitoring genetic transformation [24,25 ]. Green Fluorescent Protein (GFP) is a Green fluorescent-emitting protein obtained from victoria multitubular luminescent jellyfish (Aequoreavictoria), and its gene can be expressed heterologously in other species. Through continuous mutation and screening, the fluorescent protein species which are more suitable for application in various aspects such as exciting light wavelength, emission light wavelength, fluorescence intensity and the like are gradually obtained. The results of the study showed that GFP is not toxic to the cells and does not require substrates or cofactors. The observation of genetic transformation is simple and real-time since GFP can be directly observed without the need for exogenous substrates. The advantages of GFP can be used to realize the localization of target protein in tissues and cells, or to analyze and sort cells through green fluorescent protein, while the classical drug screening not only needs to use drugs and takes a long time (weeks or even months) to eliminate negative populations to obtain high-yield cells. In addition, the drug treatment can cause great damage to the target high-yield cells. The use of GFP as a screening marker or reporter system requires only observation and physical separation or screening by flow cytometry, thus enabling positive cells to be obtained rapidly and efficiently from a large population of negative cells [24-26] without substantial damage to the cells.

In the invention, the green fluorescent protein gene has a nucleotide sequence shown as SEQ ID NO.5 and a variant thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 5. The difference is caused by substitution, deletion or addition of a base.

In the invention, the green fluorescent protein has an amino acid sequence shown as SEQ ID NO.8 and a variant thereof. The variant has a nucleotide sequence having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID No. 8. The difference is caused by substitution, deletion or addition of amino acids.

It is to be understood that the present invention is not limited to the green fluorescent protein gene, and those skilled in the art can select other suitable fluorescent genes as needed and are within the scope of the present invention.

In some embodiments, the expression vectors of the invention further comprise other regulatory control elements.

The elements that drive expression of the protein product of interest are referred to as "regulatory control elements," which include transcriptional activators and enhancers, transcriptional initiation and termination elements, translational initiation and termination elements, and secretion signal leaders, among others. Any optimization must first be done independently for each of these elements. Once the individual elements are optimized, they must be assembled and tested to ensure that the assembly of a given element is compatible and capable of directing efficient expression of the desired recombinant protein product.

Cis-elements including promoters, enhancers, distal enhancers, etc. upstream of the albumin gene have been identified in detail. These elements include promoter and enhancer elements near the first intron, an enhancer 1.7kb upstream of the transcription start site, and a distal enhancer 6-7kb upstream. Upstream to the further distal of the gene, there are some elements that regulate negatively [7,8 ]. The albumin gene promoter and its adjacent binding sites containing TATA box, liver nuclear factor 1 (HNF 1) and nuclear factor Y (NF-Y) constitute the core region for promoting albumin gene expression, and this region mainly activates albumin gene expression [7 ].

In some embodiments, the expression vector may further comprise one or more elements as described above.

It is also important that the elements be "operably linked" in the assembly of the various combinations of elements. "operably linked" refers to a functional linkage between various elements in a manner that preserves the functionality of the individual elements as well as the combined functionality of the elements, since many of the transcription and translation functions are the result of processing from one element to the next. Thus, "operably linked" means that the nucleic acid sequences of the various elements are linked and contiguous, and if desired contiguous, to maintain an appropriate reading frame for the encoded protein.

"vector" refers to a DNA molecule capable of self-replication and/or expression for transferring a DNA fragment (a target gene) to a recipient cell in a recombinant DNA technique of genetic engineering.

① in host cells can be preserved and replicated in large quantity, and it is harmless to the recipient cells, and it does not affect the normal life activity of the recipient cells, ② has multiple restriction enzymes (restriction enzymes) cut points, and it is only preferable that each cut point is one, such as pBR322 of Escherichia coli has single recognition site of multiple restriction enzymes, and it can be suitable for inserting DNA cut by multiple restriction enzymes, ③ contains replication initiation site, and can be independently replicated, and can make the recombinant DNA lost by replication, ④ has a certain marker gene, and is convenient for screening, such as pBR322 of Escherichia coli carries ampicillin resistance gene and tetracycline resistance gene, and can be used as marker gene for screening.

The vector may be a plasmid vector, a phage vector, a cosmid vector, an M13 phage vector, a phagemid vector, a virus vector, or the like. The vector of the present invention includes, but is not limited to, the above-mentioned vectors, and preferably, the vector of the present invention is a viral vector.

Plasmid and phage vectors can only be propagated in bacteria and cannot meet the eukaryotic DNA recombination requirements. Viruses that infect animals can be engineered for use as vectors for animal cells. Since culture and manipulation of animal cells are complicated and expensive, a bacterial plasmid replication initiation sequence is generally placed in the viral vector during construction. The vector and the foreign sequence carried thereby can be conveniently propagated and cloned in bacteria and then introduced into eukaryotic cells.

In the present invention, the viral vector may be selected from, for example, retroviruses, lentiviruses and adenoviruses. Preferably, the vector is a retroviral or lentiviral vector.

Lentivirus Vectors (L entiviral Vectors, L Vs) are a class of gene transfer delivery systems that have been developed depending on the infection and replication characteristics of retroviruses [27] lentivirus Vectors are designed to "dig" key viral genes (e.g., viral virion proteins and retroviral coding genes) in a lentivirus genome (e.g., HIV Virus, Humanimmunodeficiency Virus, HIV) and replace them with target genes in the viral packaging cell by supplying HIV virion proteins and retroviral coding genes in trans in order to achieve viral particle assembly in the viral packaging cell.

Although lentiviral vectors are provided in the examples of the present invention, it is understood that those skilled in the art can select appropriate viral vectors according to circumstances to achieve the objects of the present invention, and it is within the scope of the present invention.

In a second aspect of the invention, the invention provides an expression cell comprising an expression vector as described above. In the present invention, the expression cell is a mammalian expression cell.

As used herein, the term "mammal" includes any human or non-human mammal, including, but not limited to, porcines, sheep, bovines, rodents, ungulates, pigs, sheep, lambs, goats, cattle, deer, mules, horses, primates (e.g., monkeys), dogs, cats, rats, and mice. The term "cell" includes any cell, such as, but not limited to, any human or non-human mammalian cell described herein. The cells may be conventional cells or non-conventional cells (e.g., transformed cells, established cells, or cells derived from a diseased tissue sample). The cell may be a somatic cell, such as a fibroblast or a keratinocyte. Preferred cells are stem cells such as, but not limited to, embryonic stem cells, fetal stem cells, adult stem cells, and pluripotent stem cells such as induced pluripotent stem cells. Particularly preferred cells are human embryonic stem cells, human fetal stem cells, human adult stem cells, and human pluripotent stem cells such as induced human pluripotent stem cells.

In an embodiment of the invention, the cell is a human cell, preferably a human kidney cell or liver cell. However, it will be appreciated by those skilled in the art that the present invention is not limited to human kidney cells or liver cells.

In a third aspect of the invention, there is provided a method of producing human serum albumin, the method comprising culturing an expression cell to express human serum albumin under conditions which allow expression of the human serum albumin, and recovering the human serum albumin. The conditions are well known to those skilled in the art, and the materials, reagents, equipment, and equipment used, as well as the quantities, times, temperatures, pressures, and other production parameters are well known to those skilled in the art and can be adjusted to achieve optimum production conditions.

In a fourth aspect, the present invention provides human serum albumin expressed from the expression vector or the expression cell or prepared by the method.

In a further aspect of the invention, the invention provides the use of an albumin promoter for expressing albumin, the use comprising constructing an expression vector comprising the albumin promoter. Preferably, the albumin promoter is a human serum albumin promoter and the albumin is human serum albumin.

The invention has the remarkable progress that:

(1) the carrier of the invention fills up the technical blank

One source of medicinal HSA is extracted from human blood, but due to the limited plasma volume, HSA should be in short supply and price will continuously rise, and the HSA prepared by adopting the blood source has the problems of potential diseases or virus pollution, large batch-to-batch difference and the like. The production of HSA from yeast, bacillus and rice for replacement of blood-derived albumin for clinical medicine also faces significant challenges. Thus, it is theorized that recombinant human serum albumin produced from human cells would be the best, most likely successful, replacement of blood-derived albumin for clinical use. However, no efficient expression vector for expression in human cells is currently available. The invention overcomes various difficulties and obstacles, constructs a vector for driving the expression of the exogenous gene by the promoter of the human albumin gene for the first time, successfully realizes the expression of the exogenous recombinant human albumin in human cells by using the vector and fills the technical blank. The vector also overcomes the problems of high false positive rate and expression imbalance in screening in a vector system using different promoters for the target gene and the screening marker, and has milestone contribution to the prior art.

(2) The carrier of the present invention has excellent effects

One expression vector of the two vectors of the invention takes an industrially common human glutamine synthetase gene as a screening marker, and the other vector takes green fluorescent protein as a screening marker. The high-yield cell line can be screened in mammal cells with deficiency or knock-out of glutamine synthetase by using human glutamine synthetase gene as screening marker vector, or screened by using glutamine synthetase inhibitor MSX in mammal cells with deficiency of glutamine synthetase. If the green fluorescent protein is used as a screening marker, the green fluorescent protein can be used for rapidly screening high-yield cell strains.

The carrier is utilized to successfully and efficiently express recombinant human serum albumin in human cells, the albumin secreted into a culture solution reaches 750 mg/L, and the purity exceeds 90 percent.

(3) The invention overcomes the technical problems or technical prejudice for a long time

Most preferably, albumin is serum albumin expressed by liver, accounts for 5-10% of newly synthesized liver protein, and can only be synthesized by differentiated and mature liver cells, and albumin genes in other tissue cells are in a closed state. This indicates that the albumin promoter is cell-specific, functions only in the liver, has no versatility, and is therefore not thought of as a promoter for recombinant expression vectors. Furthermore, it is known that human cells are difficult to be industrially cultured due to their specificity, and even if they can be industrially cultured, they are very expensive, and therefore cannot be industrially produced, and they are not profitable. Thus, the expression of recombinant albumin in human cells has not been contemplated by those skilled in the art, much less a successful precedent.

The invention solves the technical problem that people are eagerly to solve but never try or fail to succeed, successfully constructs a vector which drives the expression of an exogenous gene by using a promoter of a human albumin gene and successfully realizes the expression of exogenous recombinant human albumin in human cells by using the vector.

Drawings

FIG. 1 shows the sequence of the junction of the expression cassettes in the L enti801 lentiviral vector.

FIG. 2 shows the sequence of the junction of the expression cassettes in the L enti802 lentiviral vector.

FIG. 3 shows the results of detection of L enti801 in HEK293 human kidney cells for transient expression of the selection marker gene, human glutamine synthetase.

FIG. 4 is C3A nuclear GFP positive cells after viral infection.

FIG. 5 shows GFP positive stable cell lines after sorting by flow cytometry.

FIG. 6 shows the result of Coomassie blue staining of human serum albumin, a target protein expressed by stable cell lines.

FIG. 7 shows the PCR detection strategy for the presence of exogenous human serum albumin promoter and exogenous human serum albumin gene in the genome of stable cell lines.

FIG. 8 shows the results of the detection of the presence of the exogenous human serum albumin promoter and the exogenous human serum albumin gene in the genome of the stable cell line.

Detailed Description

The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.

The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.

Example 1 construction of bicistronic lentiviral vector (hA L Bpro-hA L B-IRES-hGS) L enti801 that drives expression of human serum albumin and human glutamine synthetase by human serum albumin promoter

The DNA fragment of the whole gene synthesis expression frame sequentially comprises a human serum albumin promoter (hA L Bpro) -a first intron of a human serum albumin gene-a human serum albumin gene (hA L B) -IRES-a human glutamine synthetase gene (hGS) from a5 'end to a 3' end, and is connected to a XhoI and SalI enzyme-digested vector PGK-H2BmCherry (Addgene #27217) through recombination to replace an original PGK promoter and an inserted gene of the vector.

Finally, the lentiviral vector L enti801 was obtained, see FIG. 1: L enti801 for the order of ligation of the expression cassettes in the lentiviral vector.

Example 2 human serum Albumin promoter-driven bicistronic lentiviral vector for human serum Albumin and Green fluorescent protein (hA L Bpro-hA L B-IRES-GFP) L enti802 construction

The nucleic acid fragment of the total synthetic expression frame sequentially comprises a human serum albumin promoter (hA L Bpro) -a first intron of a human serum albumin gene-a human serum albumin gene (hA L B) -IRES-Green Fluorescent Protein (GFP) from 5 'end to 3' end, is connected to a XhoI and SalI enzyme-cut vector PGK-H2BmCherry (Addge #27217) through recombination, and replaces the original PGK promoter and an inserted gene of the vector.

Finally, the lentiviral vector L enti802 was obtained, see FIG. 2: L enti802 for the order of ligation of the expression cassettes in the lentiviral vector.

Example 3 immunoblot detection of human serum Albumin transient expression in human HEK293 by L enti801 and L enti802

1. Day 1, 6 × 10 was seeded in 12-well cell culture wells⁵HEK293 or C3A cells in 1ml DMEM (GIBCO) + 10% FBS (GIBCO). Culturing in a carbon dioxide incubator at 37 deg.C for 12-16 hr.

2. On day 2, 1.5ml of the sterilized EP tube was taken, and 2. mu.g of L enti801 (or L enti802) vector DNA was added thereto, and 50. mu.l of opti-MEM was added thereto, and left to stand for 5 minutes, while in another 1.5ml of sterilized EP tube, 50. mu.l of lopti-MEM (GIBCO) was added thereto, and 6. mu.l of the lipofectamine Fugen HD (Roche) was added thereto, and left to stand for 5 minutes.

3. The two tube mixtures from step 2 were combined, the contents gently mixed with a pipette and allowed to stand at room temperature for 20 minutes.

4. The cell culture plate was removed from the incubator, the combined DNA and Fugen HD liposome solution was added dropwise to the cells in 12-well, mixed slightly and the cells were returned to the incubator for further culture.

5. On day 3, 12-16 hours later, the medium containing the transfection reagent-DNA mixture in the 12-well was replaced with well-preheated 1ml of fresh DMEM + 10% FBS, and the incubation was continued in the incubator for an additional 36 hours.

6. On day 4, 48h after transfection, the original medium was removed from the wells, the cells were washed 2 times with 1ml of fully pre-warmed serum-free RPMI1640, 1ml of serum-free DMEM was added to the wells and returned to the incubator for further incubation for 24 hours.

7. The cell culture broth was centrifuged at 1000rpm for 3min, and the culture supernatant was transferred to a 1.5ml tube. The supernatant is stored in a refrigerator at 4 ℃ for 2 weeks or stored in a refrigerator at minus 80 ℃ for a long time.

8. Mu.l of the culture supernatant was added to 2.5. mu.l of 5xSDS buffer and denatured at 90 ℃ for 3 min. After the sample is separated by the conventional SDS-PAGE electrophoresis, the human serum albumin is detected by the conventional western blotting.

Example 4L enti801 and L enti802 promote marker Gene expression in human HEK293

1. Cell transfection was accomplished according to example 3, steps 1-5. And observing whether the fluorescent protein gene is expressed or not by using a fluorescence microscope.

2. The cells were removed and the cell culture medium was aspirated. The forehead cells in the culture wells were washed with 1ml of phosphate buffer. Cells were lysed by adding 200. mu.l of 1xSDS loading buffer to the cell culture wells.

3. The cell lysate was transferred to a 1.5ml tube, centrifuged at 12000rpm for 15min, and the pellet removed. 25 μ l of lysate was taken and incubated at 90 ℃ for 3 min. After SDS-PAGE electrophoresis, the expression of the human glutamine synthetase gene was detected by western blotting technique.

The results are shown in FIG. 3, wherein L enti801 transiently expressed the results of detection of human glutamine synthetase, a selection marker gene, in human kidney cells HEK 293.

Example 5: transfection of 293T cells for lentivirus preparation

The lentivirus packaging systems used were Pax2 and VSV-G, 293T packaging cells, DMEM (GIBCO) + 10% FBS (GIBCO).

1. Day 1, 6-well cell culture wells were seeded with 1.3 × 10⁶293T cells, 2ml of cell culture medium. Culturing in a carbon dioxide incubator at 37 deg.C for 12-16 hr.

2. On day 2, 1.5ml of the sterilized EP tube was taken, and 1.6. mu.g of L enti802 (or L enti801), 1.2. mu.g of Pax2, 0.8. mu.g of VSV-G vector DNA, 100. mu.l of opti-MEM were added, and left to stand for 5 minutes, and in another 1.5ml of sterilized EP tube, 100. mu.l of lopti-MEM (GIBCO) was added, to which 10.8. mu.l of the lipofectamine Fugene HD (Roche) was added, and left to stand for 5 minutes.

4. The cell culture plate was removed from the incubator, the DNA solution and Fugen HD liposome solution were added dropwise uniformly to the medium of 293T cells in 6-well, mixed slightly, and the cells were returned to the cell culture incubator for further culture.

5. After 12-16 hours, the medium containing the transfection reagent-DNA mixture in the 6-well was replaced with a well-pre-warmed 2ml of medium and the cells were returned to the incubator for further incubation for an additional 36 hours.

6. On day 4, 48h post-transfection, virus-containing supernatants were collected. The supernatant was filtered through a 0.45 μm filter into a sterile centrifuge tube. The virus was stored in a refrigerator at 4 ℃ and used within 2 weeks.

Example 6: lentivirus infects human liver cancer cell line HepG2/C3A to establish stable cell line.

1. On day 1, cells were cultured in 10% FBS-containing RPMI1640 to logarithmic growth phase HepG2/C3A cells, which were trypsinized and counted, and inoculated with 5 × 10 cells per 24-well⁴Each cell, 500. mu. L of cell culture solution, was cultured in a cell culture incubator for 12-16 hours.

2. On day 2, the cells were removed from the incubator, 0.5. mu.l of 8mg/ml Polybrene (Sigma) was added to the cell culture medium, gently mixed, 200. mu. L of the lentivirus collected in example 5 was added to the cells, mixed, and returned to the cell incubator for further culture.

3. On day 3, the culture medium was replaced with fresh one 16 to 24 hours after infection, the culture medium containing the lentiviral particles was aspirated, and 500. mu. L DMEM + 10% FBS culture medium was added to the culture well again to continue the culture.

4. Day 4: and expanding the cells according to the conventional cell passage operation.

As shown in FIG. 4, it is C3A nuclear GFP positive cells after viral infection. As shown in FIG. 5, the GFP-positive stable cell line was obtained after sorting by flow cytometry.

Example 7: coomassie blue detection of human serum albumin in stable cell lines

1 seeding cells 6X10⁵In RPM1640+ 10% FBS in 12-well, change the medium every other day, culture until the cells overgrow the whole culture well bottom.

2 remove the medium and wash 2 times with 1ml of PBS, which was well pre-warmed. Then, 1ml of RPMI1640 medium without any addition was added thereto, and the mixture was cultured in a cell culture chamber for 24 hours.

3 taking 20 mu l of supernatant, and carrying out Coomassie brilliant blue staining on SDS-PAGE; in addition, 10. mu.l of the solution was taken for western blot detection. FIG. 6 shows the Coomassie blue staining test result of human serum albumin, a target protein expressed by stable cell strain, which confirms that the vector driven by human serum albumin promoter and used for expressing human serum albumin gene in human cell successfully and efficiently expresses exogenous recombinant human serum albumin in human hepatocyte, and simultaneously overcomes the problems of high cloning frequency and unbalanced expression of false positive cells during screening in the vector system of human serum albumin and different promoters used for screening marker.

Example 8: detection of human serum albumin gene promoter and human serum albumin CDS sequence in stable cell strain genome

In human hepatocyte genomic DNA, the human serum albumin gene a L B is present in the form of multiple exons separated by multiple introns, and the complete contiguous a L B protein coding sequence (CDS) is present only in mRNA obtained after gene transcription.

The genomic DNA of the stable cell strain obtained by examples 3 to 6 of this patent was characterized in that the promoter of the A L B gene, the first intron of A L B, and the CDS (coding sequence), IRES sequence and selection marker gene of all A L B were integrated in a continuous form at one or more positions in the cell genome.

The presence of this characteristic nucleic acid fragment can be detected by using a forward primer at the start of the A L B gene promoter and a reverse primer at the middle or end of the albumin gene, using genomic DNA from HepG2/C3A L enti802 cells as a template, and by DNA Polymerase Chain Reaction (PCR), a DNA fragment of the corresponding size can be amplified, and the DNA fragment amplified by PCR can be subjected to conventional DNA sequencing using any of the above primers, and the sequence determined is identical to (or reverse complementary to) the human serum albumin promoter and the human serum albumin gene contained in the vector L enti 802.

The specific implementation operation is as follows:

1, Collection 1 × 10⁶Extracting the genome DNA of each cell according to a conventional cell genome extraction method.

2, the extracted genomic DNA was PCR amplified by pairing primer 802Genoma L Bpro1F (SEQ ID NO.9) with primers 802Genom1451-1421R (SEQ ID NO.10) and 802Genom2421R (SEQ ID NO.11), respectively, the amplified products were separated by conventional DNA agarose gel electrophoresis to obtain 1.45kb (primers 802Genoma L Bpro1F and 802Genom1451-1421R) and 2.42kb fragments (primers 802Genoma L Bpro1F and 802Genom2421R), respectively.

PCR amplification reaction system (table 1):

TABLE 1

Genomic DNA template (rare)Released into 50ng/ul)	1 μ l (about 50 ng)
		Forward primer	0.75μl
Reverse primer	0.75μl
		dNTP	5 μ l (200 uM each)
High fidelity DNA polymerase (KOD DNA polymerase)	1μl
		2 × KOD buffer	12.5μl
H₂O	4μl
		Total volume	25μl

The procedure for PCR amplification was: see Table 2

TABLE 2

The sequences of the primers used for detection were as follows:

TABLE 3

802GenomALBpro1F(SEQ ID NO.9)	5atagtaaaaaagacacagaagccctaaaatatg3
		802Genom1451-1421R(SEQ ID NO.10)	5tacttggcaaggtccgccctgtcatcagcac3
802Genom2421R(SEQ ID NO.11)	5ttataagcctaaggcagcttgacttgcagcaacaag3

And (3) detection strategy:

the human serum albumin promoter 5 'end primer and the human serum albumin 3' end primer are adopted, and the amplification product contains 0.59kb of promoter and 1.83kb of albumin gene, which are 2.42kb in total. The 5 'end of human serum albumin promoter and the 3' end primer of albumin intermediate position are adopted, and the amplification product contains the promoter and human serum albumin N-end coding sequence, and the total length is 1.45 kb.

The detection results are shown in fig. 8, which is the detection result of detecting the presence of the exogenous human serum albumin promoter and the exogenous human serum albumin gene in the genome of the stable cell strain prepared by lentivirus infection by PCR. Lane 1 (left to right in order) is a DNA marker;

lane 2 shows the amplification product containing the promoter and albumin gene obtained by using 802Genoma L Bpro1F (SEQ ID NO.9) and 802Genom2421R (SEQ ID NO.11) as primer pairs;

lane 3 shows the amplification product containing the promoter and the N-terminal coding sequence of human serum albumin obtained by using 802Genoma L Bpro1F (SEQ ID NO.9) and 802Genom1451-1421R (SEQ ID NO.10) as primer pairs.

As can be seen, the existence of the exogenous human serum albumin promoter and the exogenous human serum albumin gene nucleotide fragment is detected in the genome of the stable cell strain.

Reference to the literature

1.Campbell,P.N.and N.E.Stone,The synthesis of serum albumin andtissue proteins in slices of rat liver and liver tumour.Biochem J,1957.66(1):p.19-31.

2.Raoufinia,R.,et al.,Overview of Albumin and Its PurificationMethods.Adv Pharm Bull,2016.6(4):p.495-507.

3.Chen,Z.,et al.,Quantitation of the residual DNA from rice-derivedrecombinant human serum albumin.Anal Biochem,2014.450:p.4-10.

4.Chen,Z.,et al.,Human serum albumin from recombinant DNA technology:challenges and strategies.Biochim Biophys Acta,2013.1830(12):p.5515-25.

5.Iseki,K.,et al.,Diverse regulations of albumin gene expression byhepatocyte growth factor in HepG2 human hepatoma cells and primary culture ofrat hepatocytes.Int J Oncol,2000.16(6):p.1141-5.

6.Liu,D.and J.Zempleni,Transcriptional regulation of the albumin genedepends on the removal of histone methylation marks by the FAD-dependentmonoamine oxidase lysine-specific demethylase 1 in HepG2 humanhepatocarcinoma cells.J Nutr,2014.144(7):p.997-1001.

7.Vorachek,W.R.,et al.,Distant enhancers stimulate the albuminpromoter through complex proximal binding sites.J Biol Chem,2000.275(37):p.29031-41.

8.Frain,M.,et al.,Binding of a liver-specific factor to the humanalbumin gene promoter and enhancer.Mol Cell Biol,1990.10(3):p.991-9.

9.Kajiyama,Y.,J.Tian,and J.Locker,Characterization of distantenhancers and promoters in the albumin-alpha-fetoprotein locus during activeand silenced expression.J Biol Chem,2006.281(40):p.30122-31.

10.Licursi,M.,et al.,In vitro and in vivo comparison of viral andcellular internal ribosome entry sites for bicistronic vector expression.GeneTher,2011.18(6):p.631-6.

11.Li,T.and J.Zhang,Stable expression of three genes from atricistronic retroviral vector containing a picornavirus and 9-nt cellularinternal ribosome entry site elements.J Virol Methods,2004.115(2):p.137-44.

12.Saleh,M.,A retroviral vector that allows efficient co-expressionof two genes and the versatility of alternate selection markers.Hum GeneTher,1997.8(8):p.979-83.

13.Ngoi,S.M.,A.C.Chien,and C.G.Lee,Exploiting internal ribosome entrysites in gene therapy vector design.Curr Gene Ther,2004.4(1):p.15-31.

14.Yeo,J.H.M.,Mariati,and Y.Yang,An IRES-Mediated Tricistronic Vectorfor Efficient Generation of Stable,High-Level Monoclonal Antibody ProducingCHO DG44 Cell Lines.Methods Mol Biol,2018.1827:p.335-349.

15.Keen,M.J.and N.T.Rapson,Development of a serum-free culture mediumfor the large scale production of recombinant protein from a Chinese hamsterovary cell line.Cytotechnology,1995.17(3):p.153-63.

16.Borth,N.,et al.,Comparison of the production of a human monoclonalantibody against HIV-1 by heterohybridoma cells and recombinant CHO cells:Aflow cytometric study.Cytotechnology,1996.22(1-3):p.129-38.

17.Enosawa,S.,et al.,Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of abioartificial liver.Cell Transplant,2000.9(5):p.711-5.

18.Ng,S.K.,Generation of high-expressing cells by methotrexateamplification of destabilized dihydrofolate reductase selectionmarker.Methods Mol Biol,2012.801:p.161-72.

19.Fan,L.,et al.,Improving the efficiency of CHO cell line generationusing glutamine synthetase gene knockout cells.Biotechnol Bioeng,2012.109(4):p.1007-15.

20.Noh,S.M.,S.Shin,and G.M.Lee,Comprehensive characterization ofglutamine synthetase-mediated selection for the establishment of recombinantCHO cells producing monoclonal antibodies.Sci Rep,2018.8(1):p.5361.

21.Tang,N.H.,et al.,Ammonia metabolism capacity of HepG2 cells withhigh expression of human glutamine synthetase.Hepatobiliary Pancreat Dis Int,2008.7(6):p.621-7.

22.Xu,P.,et al.,Effects of glutamine and asparagine on recombinantantibody production using CHO-GS cell lines.Biotechnol Prog,2014.30(6):p.1457-68.

23.Tang,N.,et al.,Stable overexpression of arginase I and ornithinetranscarbamylase in HepG2 cells improves its ammonia detoxification.J CellBiochem,2012.113(2):p.518-27.

24.Meng,Y.G.,et al.,Green fluorescent protein as a second selectablemarker for selection of high producing clones from transfected CHOcells.Gene,2000.242(1-2):p.201-7.

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Sequence listing

<110> Shanghai Ann Biotechnology Ltd

<120> Albumin expression vector driven by Albumin promoter

<130>L18100174F

<160>11

<170>SIPOSequenceListing 1.0

<210>1

<211>579

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>1

atagtaaaaa agacacagaa gccctaaaat atgtatgtat gtatatgtgt gtgtgcatgc 60

gtgagtactt gtgtgtaaat ttttcattat ctataggtaa aagcacactt ggaattagca 120

atagatgcaa tttgggactt aactctttca gtatgtctta tttctaagca aagtatttag 180

tttggttagt aattactaaa cactgagaac taaattgcaa acaccaagaa ctaaaatgtt 240

caagtgggaa attacagtta aataccatgg taatgaataa aaggtacaaa tcgtttaaac 300

tcttatgtaa aatttgataa gatgttttac acaactttaa tacattgaca aggtcttgtg 360

gagaaaacag ttccagatgg taaatataca caagggattt agtcaaacaa ttttttggca 420

agaatattat gaattttgta atcggttggc agccaatgaa atacaaagat gagtctagtt 480

aataatctac aattattggt taaagaagta tattagtgct aatttccctc cgtttgtcct 540

agcttttctc ttctgtcaac cccacacgcc tttggcaca 579

<210>2

<211>1830

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60

gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120

gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180

gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240

gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300

gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360

gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420

agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480

aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540

tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600

tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660

agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720

gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780

gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840

agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900

gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960

gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020

aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080

aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140

ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200

tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260

cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320

caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380

tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440

ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500

tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560

tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620

tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680

cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740

aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800

gctgcaagtc aagctgcctt aggcttataa 1830

<210>3

<211>587

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>3

gccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc 60

ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 120

cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 180

aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 240

gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 300

gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 360

gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 420

acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 480

ggtacacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc 540

acggggacgt ggttttcctt tgaaaaacac gatgataata tggccac 587

<210>4

<211>1122

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>4

atgaccacct cagcaagttc ccacttaaat aaaggcatca agcaggtgta catgtccctg 60

cctcagggtg agaaagtcca ggccatgtat atctggatcg atggtactgg agaaggactg 120

cgctgcaaga cccggaccct ggacagtgag cccaagtgtg tggaagagtt gcctgagtgg 180

aatttcgatg gctctagtac tttacagtct gagggttcca acagtgacat gtatctcgtg 240

cctgctgcca tgtttcggga ccccttccgt aaggacccta acaagctggt gttatgtgaa 300

gttttcaagt acaatcgaag gcctgcagag accaatttga ggcacacctg taaacggata 360

atggacatgg tgagcaacca gcacccctgg tttggcatgg agcaggagta taccctcatg 420

gggacagatg ggcacccctt tggttggcct tccaacggct tcccagggcc ccagggtcca 480

tattactgtg gtgtgggagc agacagagcc tatggcaggg acatcgtgga ggcccattac 540

cgggcctgct tgtatgctgg agtcaagatt gcggggacta atgccgaggt catgcctgcc 600

cagtgggaat ttcagattgg accttgtgaa ggaatcagca tgggagatca tctctgggtg 660

gcccgtttca tcttgcatcg tgtgtgtgaa gactttggag tgatagcaac ctttgatcct 720

aagcccattc ctgggaactg gaatggtgca ggctgccata ccaacttcag caccaaggcc 780

atgcgggagg agaatggtct gaagtacatc gaggaggcca ttgagaaact aagcaagcgg 840

caccagtacc acatccgtgc ctatgatccc aagggaggcc tggacaatgc ccgacgtcta 900

actggattcc atgaaacctc caacatcaac gacttttctg ctggtgtagc caatcgtagc 960

gccagcatac gcattccccg gactgttggc caggagaaga agggttactt tgaagatcgt 1020

cgcccctctg ccaactgcga ccccttttcg gtgacagaag ccctcatccg cacgtgtctt 1080

ctcaatgaaa ccggcgatga gcccttccag tacaaaaatt aa 1122

<210>5

<211>720

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>5

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210>6

<211>609

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>6

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

35 40 45

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 5560

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

405 410 415

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

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

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly

595 600 605

Leu

<210>7

<211>373

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>7

Met Thr Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Val

1 5 10 15

Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp

20 25 30

Ile Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp

35 40 45

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

50 55 60

Ser Ser Thr Leu Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Val

65 70 75 80

Pro Ala Ala Met Phe Arg Asp Pro Phe Arg Lys Asp Pro Asn Lys Leu

85 90 95

Val Leu Cys Glu Val Phe Lys Tyr Asn Arg Arg Pro Ala Glu Thr Asn

100 105 110

Leu Arg His Thr Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His

115 120 125

Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly

130 135 140

His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro

145 150 155 160

Tyr Tyr Cys Gly Val Gly Ala Asp Arg Ala Tyr Gly Arg Asp Ile Val

165 170 175

Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Ala Gly

180 185 190

Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro

195 200 205

Cys Glu Gly Ile Ser Met Gly Asp His Leu Trp Val Ala Arg Phe Ile

210 215 220

Leu His Arg Val Cys Glu Asp Phe Gly Val IleAla Thr Phe Asp Pro

225 230 235 240

Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe

245 250 255

Ser Thr Lys Ala Met Arg Glu Glu Asn Gly Leu Lys Tyr Ile Glu Glu

260 265 270

Ala Ile Glu Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr

275 280 285

Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His

290 295 300

Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Ser

305 310 315 320

Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr

325 330 335

Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys Asp Pro Phe Ser Val Thr

340 345 350

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

355 360 365

Phe Gln Tyr Lys Asn

370

<210>8

<211>239

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>8

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

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

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210>9

<211>33

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>9

atagtaaaaa agacacagaa gccctaaaat atg 33

<210>10

<211>31

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>10

tacttggcaa ggtccgccct gtcatcagca c 31

<210>11

<211>36

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>11

ttataagcct aaggcagctt gacttgcagc aacaag 36

Claims

1. The expression cell is characterized by being a human hepatocyte, comprising an expression vector driven by an albumin promoter, wherein the expression vector is a lentiviral vector L enti801 and/or L enti 802;

the lentiviral vector L enti801 comprises a DNA fragment of a total gene synthesis expression frame, wherein the expression frame fragment sequentially comprises a human serum albumin promoter, a first intron of the human serum albumin gene, a human serum albumin gene (hA L B), an IRES (IRES), and a human glutamine synthetase gene (hGS) from a5 'end to a 3' end, and the fragment is connected to a XhoI and SalI enzyme-digested vector PGK-H2BmCherry through recombination to replace the original PGK promoter, H2B gene and mCherry gene of the vector;

the lentiviral vector L enti802 comprises a nucleic acid fragment of a total synthesis expression frame, the fragment sequentially comprises a human serum albumin promoter, a first intron of a human serum albumin gene, a human serum albumin gene (hA L B), an IRES, Green Fluorescent Protein (GFP) from a5 'end to a 3' end, and the fragment is connected to the XhoI and SalI enzyme-digested vector PGK-H2BmCherry through recombination to replace the original PGK promoter, H2B gene and mCherry gene of the vector;

the human serum albumin promoter and the first intron have nucleotide sequences shown in SEQ ID NO.1, the human serum albumin gene has a nucleotide sequence shown in SEQ ID NO.2, and the IRES has a nucleotide sequence shown in SEQ ID NO. 3.

2. The expression cell of claim 1, wherein: the human glutamine synthetase gene has a nucleotide sequence shown by SEQ ID NO.4, and the green fluorescent protein gene has a nucleotide sequence shown by SEQ ID NO. 5.

3. A method of producing human serum albumin, comprising: the method comprising culturing the expression cell of any one of claims 1-2 under conditions that allow expression of human serum albumin, and recovering the human serum albumin.