CN109504709A - The albumin expression vectors of albumin promoter driving - Google Patents

Abstract

The invention discloses what is driven by human serum albumin promoter to be used to express the carrier of albumin gene in cell, and the high efficient expression exogenous albumin that succeeds in cell.The expression vector is made of skeleton and the expression cassette being loaded on skeleton, which is made of human serum albumins promoter and its First Intron, albumin gene, internal ribosome entry site sequence (IRES) and marker gene.Human serum albumin promoter and IRES sequence driving albumin gene and marker gene are expressed simultaneously in the carrier.Using the carrier, success efficiently expressing recombinant human seralbumin, the albumin being secreted into culture solution reach 750mg/L in people's cell, and purity is more than 90%.The carrier is the carrier that the first employment blood albumin promoter drives human serum albumin gene expression, and the first carrier for being used for people's cell or mammalian cell expression albumin.The carrier also overcomes high false positive rate and the unbalanced problem of expression in the carrier system of target gene and selection markers point different promoters in screening.

Description

The albumin expression vectors of albumin promoter driving

Technical field

The invention belongs to gene engineering technology fields, the expression vector of albumin promoter driving are disclosed, specifically, public The expression vector for the sero-abluminous human serum albumins promoter driving of efficiently expressing recombinant human in people's cell is opened, more Specifically, pair of human serum albumins promoter driving external source human serum albumin gene and selection markers while expression is disclosed Cistron carrier and its sero-abluminous application of efficiently expressing recombinant human in people's cell.

Technical background

Human serum albumins (Human Serum Albumin, HSA) is that the main, content in blood of human body is most abundant Soluble protein, content accounts about the half [1] of total serum protein.The main physiological function of human serum albumins includes: 1, increase blood volume and maintains plasma colloid osmotic pressure；Adjust the dynamic equilibrium of moisture between tissue and blood vessel；2, it transports and solves Malicious function: albumin also can can convey different substances in conjunction with cation in conjunction with anion, including fatty acid, cholesterol, Noxious material can also be transported to removing toxic substances organ by thyroid hormone etc.；3, nutrition supply: histone and plasma protein can be mutual Inversion of phases, in nitrogen metabolism obstacle, it is tissue with nutrient that albumin, which can be used as nitrogen source,.Human serum albumins is in clinical medicine Use had more than 70 years.It primarily can be used for: 1, it suffers a shock caused by blood loss wound, burn；2, brain edema and damage are drawn The cranium pressure risen increases；3, oedema or ascites caused by cirrhosis and nephrosis；4, the prevention and treatment of Hypoproteinemia；5, newborn is high, and gallbladder is red Plain mass formed by blood stasis；6, for cardiopulmonary bypass, the adjuvant treatment of burn, the adjuvant treatment of haemodialysis and adult respiratory distress syndrome (ARDS) Deng.

Medicinal HSA extracts [2] from people's blood blood.But cause HSA for not due to adopting the limitation of slurry amount in China It should ask, price will persistently rise.Using blood source preparation HSA there is also blood source there may be potential disease or virus pollution, The problems such as difference is big between batch.Therefore by the strategy that the method for genetic engineering produces HSA become focus in industry and Trend.By DNA recombinant technique, HSA is produced from yeast and rice and has been achieved for huge success [3], portioned product is [4] are ratified in the clinical trial for being obtained government's drug administration department.But above-mentioned recombination HSA is used as clinic for replacement blood source albumin Drug still faces huge challenge.Due to the intracorporal critical function molecule of human serum albumins system people, theoretically, generated from people's cell Recombination human serum albumin will to be that best, most probable successfully replaces blood source albumin to be used for clinical.But it there is no at present Efficient expression vector for being expressed in people's cell.

Human serum albumins is mainly generated by liver, and there are also expression [1,2] for kidney.Human serum albumin gene is being developed During show and gradually rise, the characteristics of very stably expressing is presented after adult in liver.In specific physiology and disease Under conditions of reason, Expression of Albumin can generate certain response to environmental changes such as nutrition, pressure locating for cell and occur Change.There are promoter and multiple positively and negatively cis-regulating elements, these yuan for regulatory region in albumin gene upstream The trans-acting factors interaction such as part and intracellular a variety of different signal transduction molecules, to adjust the expression of albumin [5,6]。

In mouse, albumin gene upstream includes that the cis elements such as promoter, enhancer, Distal enhancer are detailed Thin identifies.These elements include the promoter and enhancer element close to First Intron, on transcription initiation site There is an enhancer at trip 1.7kb, there are one Distal enhancers in the place of 6-7kb in upstream.Upstream region of gene to more distal end, Then there is the element [7,8] of some negative regulations.By albumin gene promoter and its close on containing TATA box, hepatocyte nuclear factor (hepatic nuclear factor 1, HNF1) and nuclear factor Y (nuclear factor Y, NF-Y) binding site, composition The nucleus of starting albumin gene expression, this region is mainly to activate based on the expression of white protein-2 genes [7].Due to white Protein gene is stable in liver cell, lasting and high-caliber expression.Therefore should may be comprising the region of albumin promoter Ideal promoter [7,9] of foreign gene when stability and high efficiency is persistently expressed in people's cell.In the present invention, applicant constructs white The expression vector of protein promoter driving is used for the high level expression human seralbumin egg in mammalian cell, especially people's cell It is white.

Summary of the invention

It is an object of the present invention to provide a kind of expression vectors of albumin promoter driving, express people using the carrier Seralbumin.

A further object of the present invention is to provide a kind of expression cell, which can express human serum albumins.

A further object of the present invention is to provide a kind of method for producing human serum albumins.

It is also another object of the present invention to provide the applications of human serum albumins promoter.

To achieve the goals above, The technical solution adopted by the invention is as follows:

In one aspect, the present invention provides a kind of expression vectors of albumin promoter driving, it is characterised in that: described Expression vector includes expression cassette, the expression cassette successively include albumin promoter and its First Intron, albumin gene, Internal ribosome entry site sequence IRES, the second gene.

As used herein, following term and phrase should have meaning described below.Unless otherwise defined, All technologies used herein and scientific term, which all have, usually understands identical meaning with those skilled in the art.

Unless be in addition explicitly indicated herein, otherwise singular " one " (kind /) and "the" or " described includes a plurality of fingers Show object.

For term "comprising" to forgive the use of sexual openness meaning, this means to may include other elements, element.

In the present invention, " expression cassette " means that promoter element adjusts control element with other transcription and translations effectively connecting Combination.Heterologous gene sequence can be inserted into the purpose that expressing said gene sequence is used in expression cassette.Expression cassette energy Enough guidance transcriptions, the transcription cause to generate the mRNA about required gene product.Expression cassette is inserted into carrier with generation table Up to carrier, expression of such expression vector guidance of heterologous protein matter in host cell.

" promoter " is made of two essential parts (core promoter and proximal promoter).Promoter is located at given gene The upstream of coded sequence.Core promoter is defined as that the minimum nucleotides sequence of the accurate transcription starting of given gene can be instructed Column.Core promoter in eucaryote is responsible for the starting that guidance passes through rna plymerase ii compound.Generally by core promoter It is portrayed as the sequence across transcription initiation site (INR), more specifically the sequence of 5 to 45 nucleotide of INR upstream and downstream (70-90 nucleotide of total length).

In the present invention, using " albumin promoter ", preferably human serum albumins promoter.Human seralbumin Albumen is mainly generated by liver, and there are also expression [1,2] for kidney.Human serum albumin gene showed during development by Edge up height, and the characteristics of very stably expressing is presented in liver after adult.Under conditions of specific physiology and pathology, albumin Expression can generate certain response to environmental changes such as nutrition, pressure locating for cell and change.In albumin gene There are promoter and multiple positively and negatively cis-regulating elements, these elements and into the cell a variety of differences for the regulatory region of upstream The trans-acting factors interaction such as signal transduction molecule, to adjust the expression [5,6] of albumin.

In mouse, albumin gene upstream includes that the cis elements such as promoter, enhancer, Distal enhancer are detailed Thin identifies.These elements include the promoter and enhancer element close to First Intron, on transcription initiation site There is an enhancer at trip 1.7kb, there are one Distal enhancers in the place of 6-7kb in upstream.Upstream region of gene to more distal end, Then there is the element [7,8] of some negative regulations.By albumin gene promoter and its close on containing TATA box, hepatocyte nuclear factor (hepatic nuclear factor 1, HNF1) and nuclear factor Y (nuclear factor Y, NF-Y) binding site, composition The nucleus of starting albumin gene expression, this region is mainly to activate based on the expression of white protein-2 genes [7].Due to white Protein gene is stable in liver cell, lasting and high-caliber expression.Therefore should may be comprising the region of albumin promoter Ideal promoter [7,9] of foreign gene when stability and high efficiency is persistently expressed in people's cell.

In addition, other seralbumin promoters in addition to human serum albumins promoter can be used for it is of the invention Design, not departing from protection scope of the present invention.

In the present invention, human serum albumins promoter and its First Intron have as shown in SEQ ID NO.1 Nucleotide sequence and its variant.The variant and SEQ ID NO:1 have at least 85% (for example, at least 85%, at least 90%, until Few 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%) nucleotide sequence of sequence identity.The difference replaces, misses or adds institute by base It causes.

" albumin " is naturally occurring protein in mammalian plasma, and wherein it is the highest protein of abundance.It The required osmotic pressure of blood is being maintained, and is in addition being played an important role in the transport of various substances in blood flow.To take pride in The albumin of several species has been characterized, they enjoy the sequence and structural homology of height." seralbumin ", i.e., Serum albumin synthesizes in liver, is the most abundant protein of content in vertebrate blood plasma.The serum of separate sources is white The amino acid sequence and its space structure of albumen are very conservative, it has the property for combining and transporting endogenous and exogenous material Matter maintains blood colloid osmotic pressure, removes free radical, inhibits the physiological functions such as platelet aggregation and anticoagulation.

In the present invention, albumin can be selected from human albumin, non-human primates albumin (the white egg of such as chimpanzee White, gorilla albumin or the albumin of macaque, rodent albumin (such as albumin of hamster albumin, cavy, small The albumin of mouse and the albumin of rat), the albumin such as horse of the albumin (such as milk bovine albumin) of Bovidae, equine it is white Albumen or the albumin of donkey, the albumin of Canidae, the albumin of cat family, the albumin of rabbit, the albumin of goat, sheep white egg The albumin of white, chicken albumin and pig.The albumin of particularly preferred mature form, and those skilled in the art are able to use public affairs It opens available information such as Protein Data Bank and/or maturation is identified by using signal peptide identification software such as SignalP Form.It is preferred that SignalP4.0 editions.

Preferably, albumin of the invention is human serum albumins.However, it is to be appreciated that the present invention is not limited to human serums Albumin, the albumin from other animals are also applied for the present invention, within the protection scope that the present invention covers.

The single chain protein matter that human serum albumins is made of 585 amino acid, molecular weight 67kDa.Mature people's blood Pure albumen is one heart-shaped molecule, is made of the similar α-helixstructure domain of 3 structures, its concentration is about 42g/ in blood plasma L accounts for about the 60% of Total plasma protein.Human serum albumins can be solid with transport of fatty acids, BILE PIGMENTS, amino acid, class in body fluid Alcohol hormone, metal ion and many treatment molecules etc.: while maintaining the normal osmotic pressure of blood.Clinically human serum albumins Can be used for treating shock and burn, for supplementing because of operation, contingency or big bleeding caused by blood loss, can also be used as Plasma extender.

Preferably, human serum albumin gene of the invention has nucleotide sequence shown in SEQ ID NO.2 or its change Body sequence, human serum albumins of the invention have amino acid sequence or its variant sequence thereof shown in SEQ ID NO.6.

Term " variant " refers to includes at one or more (several) positions in parent's albumin gene or albumin The gene or polypeptide for changing and (replacing, be inserted into, and/or lack).Substitution, which refers to different base or amino acid, replaces original Come the base or amino acid to plant oneself；Missing refers to the base or amino acid that removal planted oneself originally；And it is inserted into and refers to increasing Add new base or amino acid.

In the present invention, it is preferred to variant human serum albumin gene and SEQ ID NO:2 have at least 85% (for example, 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%) nucleotide sequence of sequence identity.The difference is by base Caused by replacing, missing or adding.

In the present invention, it is preferred to which variant human serum albumins and SEQ ID NO:6 are at least 85% (for example, 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%) amino acid sequence of sequence identity.The difference is taken by amino acid Caused by generation, missing or addition.

In an embodiment of the invention, expression vector of the present invention includes an albumin gene and a label base Cause constitutes bicistronic mRNA.

The present invention is not limited to bicistronic mRNAs, and in an embodiment of the invention, expression vector of the present invention includes one The above albumin gene and/or marker gene constitute three suitable or polycistrons.

" internal ribosome entry site (internal ribosome entry site, abridge IRES) " be most early in 1988 from poliovirus (poliovirus, PV) and encephalomyocarditis virus (encephalomyocarditis Virus, EMCV) rna gene group in one section of nucleic acid sequence finding, in general, IRES is usually located at rna virus cdna group 5 ' non-translational regions (UTR).IRES can allow for mRNA machine translator from the IRES POS INT translation among mRNA, sick in this way The translation of toxalbumin can be independent of 5 ' cap sequences.In IRES sequence after being found in viral RNA, from cell mRNA In it has also been found that there are IRES, and find that these IRES are primarily present in the base for participating in cell answering pressure and other survival processes In the mRNA of cause [10].

The presence of IRES sequence can make protein translation originate the cap structure held independent of mRNA (mRNA) 5 ', Make it possible directly from initiation of translation among mRNA.Thus IRES sequence provides a kind of from a mRNA while independent table It reaches and the strategy of the product of the several genes of non-fusion expression.It can design according to this feature containing several genes, contain between gene There is the polycistron expression vector of IRES, as first gene depends on 5 in bi-cistronic vectors (bicistronic mRNA) ' cap sequence, second gene then depend on IRES initiation of translation, to synthesize two albumen [10,11] from a mRNA.It is logical The expression of two albumen before and after normal IRES is proportional, thus can according to the expression of one of reporter gene come Reflect the expression [12] of another gene.It can also design accordingly containing there are three gene and two IRES sequences are concatenated Polycistronic vector, the cap structure initiation of translation that first gene is held by mRNA 5 ', rear two genes are then turned over by IRES starting It translates.

This method is extremely important in most cases.Virus and non-virus carrier based on IRES have been successfully used to The common powerful of genetic manipulation [13,14] in preclinical and clinical polygene combined treatment and basic research.It is common Mode be purpose gene depend on 5 ' cap sequence initiation of translation, and selection markers depend on IRES initiation of translation.It is based on The expression strategy of the carrier of IRES is substantially better than two genes and the strategy that different promoters is expressed is respectively adopted.At two When promoter respectively drives target gene and screening-gene expression, since there are the competitions between different promoters, it is possible to cause Alternative one is by the expression of selectivity or a gene expression amount height, and another is not expressed then or expression quantity is low.IRES Carrier then can be realized target gene and riddled basins and be expressed with metastable intensity.

The large-scale production of therapeutic recombinant proteins drug (such as recombinant monoclonal antibodies or enzyme) be usually all It is realized in Chinese hamster ovary cell (Chinese hamster ovary, CHO).By being imported into CHO cell genome Expression cassette comprising target gene and riddled basins, and filter out from cell mass stablize insert containing target gene and The cell of the expression cassette of riddled basins, to establish the cell strain [15,16] of stable express express target protein.From being largely free of Stable and high level expression destination protein cell strain is obtained in the cell colony of destination gene expression frame, depends on selection markers The selection of gene and effective screening technique.Industrially-the MTX based on dihyrofolate reductase screens (Dihydrofolate Reductase (DHFR)-based methotrexate (MTX) selection) or based on glutamine synthelase (glutamine synthetase)-MSX screens [17] (GS-based methionine sulfoximine (MSX) It selection) is two kinds of main screening systems used at present.Two kinds of methods cut both ways, comparatively GS-MSX screening system It unites more more advanced.DHFR screening system uses extensively because it can obtain high expression level by gene magnification It is general.But its defect is the more wheel MTX screening needed by various concentration, thus it is time-consuming very long [18].GS screening system only needs One wheel MSX screening, therefore save the time.The system for relying on GS is carried out in addition to that can be not required to MSX in the CHO of GS-knockout Except screening, [19] can also be screened using MSX in the cell knocked out without GS.In addition, since GS can be by cell The ammonia generated when being metabolized amino acid is re-converted into glutamine, is accumulated to eliminate accumulation and elimination of the ammonia in culture solution by ammonia Tired bring pH changes and toxicity, therefore to cell growth band positive influence [20].Thus, glutamine synthelase screening System is the screening system [21-23] for having more advantage.

In the present invention, IRES sequence has nucleotide sequence and its variant as shown in SEQ ID NO.3.The variant With SEQ ID NO:3 have at least 85% (for example, 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 Nucleotide sequence.The difference is caused by the the replacing, missing or adding of amino acid.

In one embodiment, expression vector of the invention includes an IRES.In another embodiment, this hair Bright includes two or more IRES, thus constitutes three suitable or polycistrons.

" marker gene " is the exclusive noun of genetic engineering originally, but it has become a kind of basic experiment now Tool is widely used in the research of molecular biology, cell biology, Developmental Biology etc..Marker gene be it is a kind of Know the gene of function or known array, specific marker can be played a part of, judge mark gene is capable of based on its activity Whether expression.For in genetic engineering meaning, it is the vital signs of recombinant DNA carrier, is converted successfully commonly used to examine Whether；For in assignment of genes gene mapping meaning, it is the tool indicated to target gene, is existed commonly used to testing goal gene Positioning in cell.Active detection, can be directly detection label protein activity its own, can also be by via color The metabolin that this kind of activity because of label protein of element occurs is detected indirectly.Detection can be chemical detection (including enzyme The detection of reaction), the organoleptic detection of physical detection (detection including behavioural analysis) or tester (including the use of vision, tactile, Smell, the sense of hearing, the detection of the sense of taste) any one.

In the present invention, as long as marker gene or the type of label protein can be examined using well known method Its activity is surveyed just to be not particularly limited.The host for possessing transformant judge mark object when preferably detecting, i.e., for transformant The low label protein of invasion.For example, it may be drug resistance albumen, fluorescence protein, pigment synthesis protein, luminous egg White matter, external secretion protein, the protein for controlling formalness etc..It is by drug resistance gene and fluorescent marker gene Common and effective screening technique.

In some embodiments of invention, marker gene is drug resistance gene, it is preferable that drug labelling gene is selected from people Glutamine synthetase gene, kalamycin resistance gene (nptII), tetracycline resistance gene (tetR), neomycin phosphoric acid turn Move enzyme gene (npt), puromycin-resistant base, it is preferable that be people's glutamine synthetase gene.

In the present invention, GenBank AAB30693's gene have the nucleotide sequence as shown in SEQ ID NO.4 and its Variant.The variant and SEQ ID NO:4 have at least 85% (for example, 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%) nucleotide sequence of sequence identity.The difference is caused by the the replacing, missing or adding of base.

In the present invention, GenBank AAB30693 has amino acid sequence and its variant as shown in SEQ ID NO.7. The variant and SEQ ID NO:7 have at least 85% (for example, 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 is same The nucleotide sequence of one property.The difference is caused by the the replacing, missing or adding of amino acid.

It should be understood that those skilled in the art can be according to need the present invention is not limited to GenBank AAB30693's gene Other suitable drug resistance genes are selected, and within protection scope of the present invention.

In certain embodiments of the present invention, marker gene is fluorescent marker gene, it is preferable that is green fluorescent protein Or red fluorescent protein.

Green fluorescent protein or red fluorescent protein are visual selection marks that is important and being widely used in monitoring genetic transformation Remember [24,25].Green fluorescent protein (Green fluorescent protein, GFP) is from Victoria's multitube luminescent jellyfish (Aequoreavictoria) what is obtained in has the albumen of transmitting green fluorescence, and gene can carry out different in other species Source expression.By being constantly mutated and screening, gradually obtain in various aspects such as excitation wavelength, wavelength of transmitted light, fluorescence intensities It is more suitable the fluorescin type of application.Result of study show GFP there is no toxicity for cell, do not need substrate or auxiliary because Son.Since GFP can be observed directly without xenobiotic substrates, so that the observation of genetic transformation is simply and real-time.Utilize the excellent of GFP Point may be implemented positioning to destination protein in tissue and cell, or by green fluorescent protein to cell implement analysis and Sorting, and classical drug screening not only needs to take a long time (even some months several weeks) using drug to eliminate feminine gender Group and obtain high yield cell.In addition drug-treated also can bring biggish damage to purpose high yield cell.Use GFP as sieve Choosing label or reporting system only need to be screened by observation and physical separation or flow cytometer, so as to from a large amount of Negative cell populations in acquisition positive cell [24-26] rapidly and efficiently, damage will not be generated to cell substantially.

In the present invention, green fluorescence protein gene has nucleotide sequence and its variant as shown in SEQ ID NO.5. The variant and SEQ ID NO:5 have at least 85% (for example, 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 is same The nucleotide sequence of one property.The difference is caused by the the replacing, missing or adding of base.

In the present invention, green fluorescent protein has amino acid sequence and its variant as shown in SEQ ID NO.8.The change Body and SEQ ID NO:8 have at least 85% (for example, 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 is same The nucleotide sequence of one property.The difference is caused by the the replacing, missing or adding of amino acid.

It should be understood that those skilled in the art can select as needed the present invention is not limited to green fluorescence protein gene Other suitable fluorogenes are selected, and within protection scope of the present invention.

In some embodiments, expression vector of the invention also includes that other adjust control element.

The element of target protein product expression is driven to be known as " adjusting control element ", adjusting control element includes that transcription swashs Son living and enhancer, transcription initiation and termination element, translation initiation and termination element and secretion signal leader sequence etc.. Any optimization must be carried out independently first for each thing in these elements.Once element is optimized alone, it Just must be assembled and be tested, to ensure that the assembly of point element is compatible and can instruct required recombinant protein The effective expression of product.

Albumin gene upstream includes the cis elements such as promoter, enhancer, Distal enhancer by detailed identification Out.These elements include the promoter and enhancer element close to First Intron, at the 1.7kb of transcription initiation site upstream There is an enhancer, there are one Distal enhancers in the place of 6-7kb in upstream.In upstream region of gene to more distal end, then have one The element [7,8] of a little negative regulations.By albumin gene promoter and its close on containing TATA box, hepatocyte nuclear factor (hepatic Nuclear factor 1, HNF1) and nuclear factor Y (nuclear factor Y, NF-Y) binding site, constitute the white egg of starting The nucleus of white gene expression, this region is mainly to activate based on the expression of white protein-2 genes [7].

In some embodiments, expression vector can also include one or more elements as described above.

In the various combined assembly of element, it is also important that these elements are " effectively to connect (operably linked)"." effectively connecting " refers to the functional connection between various elements in this way, and the mode remains The function of the function of each element and combined element, because many transcription and translation functions are from an element to next The result that element is handled.Therefore, it " effectively connects " it is meant that the nucleic acid sequence of various elements was connected to connect and abut, And the reading frame being adjacently attached when needed to keep suitable coding protein.

" carrier (vector) ", which refers to, is transferred to receptor for DNA fragmentation (target gene) in genetic engineering recombinant DNA technology Cell it is a kind of can self-replacation and/or expression DNA molecular.

The necessary condition of carrier: 1. can preserve in host cell and energy massive duplication, and harmless to recipient cell, The normal vital movement of recipient cell is not influenced.2. having the point of contact multiple restriction enzymes (restriction enzymes), and every kind The point of contact of enzyme preferably only one, such as Escherichia coli pBR322 if there are many restriction enzyme single recognition site, may be adapted to a variety of The DNA insertion of restriction enzyme cutting.3., can be independently duplicated containing replication origin；Gene magnification is carried out by duplication, otherwise Recombinant DNA may be made to lose.4. there is certain marker gene, it is convenient for screening.As the pBR322 plasmid of Escherichia coli is taken Band ampicillin resistance gene and tetracycline resistance gene, so that it may marker gene as a filter.In general, natural to carry Body tends not to meet above-mentioned requirements, it is therefore desirable to according to different purpose and needs, manually be reconstructed to carrier.Now Used plasmid vector is nearly all by reconstruction.5. vector dna molecule size should be suitable, to operate.

The type of carrier has plasmid vector, phage vector, coemid carrier, M13 phage vector, phasmid to carry Body, viral vectors etc..Carrier of the invention includes but is limited to above-mentioned carrier, and preferably, carrier of the invention is viral vectors.

Plasmid and phage vector can only be bred in bacterium, not be able to satisfy eukaryotic DNA recombination needs.The disease of infection animal The carrier as zooblast can be transformed in poison.Since the culture and operation of zooblast are more complex, it is also more to spend, thus virus Generally all bacterial plasmid replication initiation sequence is placed wherein when vector construction.The foreign sequence of carrier and its carrying is set to facilitate Ground is bred and is cloned in bacterium, and eukaryocyte is then re-introduced into.

In the present invention, viral vectors can be selected from such as retrovirus, slow virus and adenovirus.Preferably, carrier is Retrovirus or slow virus carrier.

Slow virus carrier (Lentiviral Vectors, LVs) is that a kind of infection for relying on retrovirus and duplication are special Property and the gene transfer transmission system [27] to grow up.Slow virus carrier is in lentiviral gene group (such as inhibition of HIV, Human Immunodeficiency Virus, HIV) in " excavate " key viral gene (such as virion albumen and reverse transcriptase coding Gene) and instead target gene.Link is generated in virus, offer inhibition of HIV virion trans- into Viral packaging cell is provided The encoding gene of albumen and reverse transcriptase, to realize that virion assembles.This virion albumen with trans- offer is realized Virion assembling be merely able to realize primary virus packaging.The virion of formation is pseudovirus, because of its genome heredity object Lack key viral virion protein gene and pol gene in matter composition.The virion of formation infect new host with It may be implemented to be integrated into host genome it includes the genomic nucleic acid fragment of target gene afterwards.Because lacking virus structure egg White, virion protein and reverse transcriptase, therefore not can be carried out the duplication of second of genome and the assembling of virion.It compares Other gene delivery systems, slow virus carrier has obvious advantage, including slow virus carrier can be irreversible whole It closes in the genomic DNA of host cell, is replicated with genome duplication, so as to realize that destination gene expression is tieed up always It holds and still contains target gene in progeny cell after certain level, Jing Duodai cell division；Slow virus titre is high, efficiency of infection It is high；By various transformations, the bearer capabilities of slow virus carrier can pack big genetic fragment；Carrier system is due to removal More viral gene and its product, immunogenicity is relatively low, highly-safe.In addition, in viral packaging system, slow virus Carrier can combine to form different pseudotype virus with many different heterologous envelope glycoproteins, realize to different type host cell Infection and cell in different states, such as proliferative cell and resting cell [27,28].Slow virus carrier is in addition to virus Except the form transfer gene of packaging, target gene can also be inserted with lower probability by efficient rotaring transfecting modes such as electricity turn Enter in the genome of aim cell.

Although providing slow virus carrier in the embodiment of the present invention, it should be appreciated that those skilled in the art are according to circumstances Suitable viral vectors can be selected, is achieved the object of the present invention, within protection scope of the present invention.

In the second aspect of the invention, the present invention provides a kind of expression cell, the expression cell includes front institute The expression vector stated.In the present invention, expression cell is mammalian expression cell.

As used herein, term " mammal " includes any mankind or non-human mammal, non-human lactation Animal includes but is not limited to Suidae, sheep, Bovidae, Rodentia, ungulate, pig, sheep, lamb, goat, ox, deer, mule, horse, spirit Long class (such as monkey), dog, cat, rat and mouse.Term " cell " includes any cell, such as, but not limited to, described herein Any mankind or non-human mammalian cell.Cell can be regular growth or unconventional cell (such as conversion cell, Establish cell or the cell derived from pathological tissues sample).Cell can be body cell, such as fibroblast or keratinocyte. Preferred cell is stem cell, and such as, but not limited to embryonic stem cell, fetal stem cell, adult stem cell and multipotential stem cell is all Such as the multipotential stem cell of induction.Particularly preferred cell is human embryo stem cell, human fetal stem cell, people's adult stem cell and people The human pluripotent stem cells that multipotential stem cell such as induces.

In embodiments of the present invention, cell is people's cell, it is preferable that is HK cells or liver cell.However, ability Field technique personnel are it should be understood that the present invention is not limited to HK cells or liver cells.

In the third aspect of the invention, the present invention provides a kind of method for producing human serum albumins, this method packets It includes under conditions of allowing human serum albumins to express, cultivates expression cell to express human serum albumins, and the recycling people Seralbumin.The condition be it is well-known to those skilled in the art, used in material, reagent, instrument, equipment, And the various manufacturing parameters such as dosage, time, temperature, pressure be all those skilled in the art it is known and can according to the actual situation into Row adjustment, to obtain optimal working condition.

In the fourth aspect of the invention, the present invention provides human serum albumins, the human serum albumins is by front The expression vector or mentioned-above expression cell are expressed to obtain, or are prepared by mentioned-above method.

In another aspect of the invention, the present invention provides application of the albumin promoter in expression albumin, should Using the expression vector for including building including albumin promoter.Preferably, the albumin promoter is human serum albumins Promoter, the albumin are human serum albumins.

The present invention has significant progress:

(1) carrier of the invention has filled up technological gap

A source of medicinal HSA is to extract from human blood, but supply falls short of demand since the limitation for adopting slurry amount leads to HSA, Price will persistently rise, and using blood source preparation HSA, there is also blood source, there may be between potential disease or virus pollution, batch The problems such as difference is big.HSA is produced from yeast, bacillus and rice is used for clinical medicine also still for replacement blood source albumin Face huge challenge.Therefore, theoretically from people's cell generate recombination human serum albumin will be it is best, most probable successfully takes For blood source albumin for clinic.But it there is no the efficient expression vector for expressing in people's cell at present.The present invention is exactly Various difficult and obstacles are overcome, are constructed for the first time with the carrier of the promoter driving exogenous gene expression of the white protein-2 genes of people's blood simultaneously It is successfully realized and expresses external source recombinant human serum albumin in people's cell using the carrier, filled up technological gap.The carrier is also Overcome the high false positive rate in the carrier system of target gene and selection markers point different promoters in screening and expression Unbalanced problem has landmark contribution to the prior art.

(2) carrier of the invention has the effect of excellent

Two kinds of carriers of the invention, a kind of expression vector is using industrial common GenBank AAB30693's gene as sieving Choosing label, another carrier is using green fluorescent protein as selection markers.Using GenBank AAB30693's gene as screening Labeled vector can carry out the screening of high yielding cell sarain in the mammalian cell of glutamine synthelase defect or knockout, or Person is sieved in the mammalian cell containing glutamine synthelase defect using the inhibitor MSX of glutamine synthelase Choosing.The green fluorescent protein that then can use using green fluorescent protein as selection markers carries out quick high yielding cell sarain sieve Choosing.

Through embodiment demonstrate this two kinds of carriers can in people's cell express express target protein (human serum albumins) and Selection markers.Using the carrier in people's cell success efficiently expressing recombinant human seralbumin, be secreted into white in culture solution Albumen reaches 750mg/L, and purity is more than 90%.Human serum albumins can be industrially mass produced in the present invention, have extensive Application value.

(3) the present invention overcomes for a long time the technical issues of or technology prejudice

First choice, albumin are the seralbumins of liver expression, account for the 5-10% of the new synthetic proteins of liver, it can only be by dividing It is melted into ripe liver cell synthesis, the albumin gene in other histocytes is in close state.This shows that albumin starts Son is cell-specific, is only worked in liver, without versatility, so not expecting opening for recombinant expression carrier by people Mover.In addition, week known to crowd, people's cell is difficult to industrialize culture due to its particularity, even if can industrialize, cost is very high It is high, thus can not industrialization, it is even more impossible to get a profit.Therefore, those skilled in the art never expect that expression recombinates white egg in people's cell It is white, more without success precedent.

The present invention solves people and thirsts for solving but dare not going always the technical problem attempted or fail to succeed always, It successfully constructs to drive the carrier of exogenous gene expression with the promoter of the white protein-2 genes of people's blood and be successfully realized and utilizes the carrier External source recombinant human serum albumin is expressed in people's cell.

Detailed description of the invention

Fig. 1 is the order of connection of expression cassette in Lenti801 slow virus carrier.

Fig. 2 is the order of connection of expression cassette in Lenti802 slow virus carrier.

Fig. 3 is inspection of the Lenti801 in HK cells HEK293 transient expression riddled basins GenBank AAB30693 Survey result.

Fig. 4 is C3A nucleus GFP positive cell after virus infection.

Fig. 5 is the GFP positive stable cell strain after selected by flow cytometry apoptosis.

Fig. 6 is destination protein-human serum albumin coomassie brilliant blue staining testing result of stable cell line expression.

Fig. 7 is that there are external source human serum albumin promoters and external source human serum albumin gene in stable cell line genome PCR inspection policies.

Fig. 8 is that there are external source human serum albumin promoters and external source human serum albumin gene in stable cell line genome Testing result.

Specific embodiment

The application is further elaborated below by detailed embodiment, it should be appreciated that following embodiments are only In order to be not defined to summary of the invention for illustrating the application.

Instrument, equipment, reagent can obtain through various channels in embodiment, such as be commercially available, or can make It is standby and obtain.

Embodiment 1: it is double suitable that human serum albumins promoter drives human serum albumin and GenBank AAB30693 to express The building of anti-sub- slow virus carrier (hALBpro-hALB-IRES-hGS) Lenti801

Full genome synthesizes the DNA fragmentation of expression cassette, which ' holds at end to 3 ': human seralbumin egg from 5 First Intron-human serum albumin gene (hALB)-IRES- of white promoter (hALBpro)-human serum albumin gene GenBank AAB30693's gene (hGS).The segment is connected to the carrier PGK- through XhoI and SalI digestion by recombination In H2BmCherry (Addgene#27217), the replacement original PGK promoter of carrier and insertion gene.

Final to obtain slow virus carrier Lenti801, the connection referring to expression cassette in Fig. 1: Lenti801 slow virus carrier is suitable Sequence.

Embodiment 2: human serum albumins promoter drives the bicistronic mRNA of human serum albumin and green fluorescent protein sick slowly Poisonous carrier (hALBpro-hALB-IRES-GFP) Lenti802 building

The nucleic acid fragment of fully synthetic expression cassette, the segment ' are held at end to 3 ': human serum albumins promoter from 5 (hALBpro)-human serum albumin gene First Intron-human serum albumin gene (hALB)-IRES- green fluorescence Albumen (GFP).The segment is connected to the carrier PGK-H2BmCherry (Addgene# through XhoI and SalI digestion by recombination 27217) in, the replacement original PGK promoter of carrier and insertion gene.

Final to obtain slow virus carrier Lenti802, referring to fig. 2: the connection of expression cassette is suitable in Lenti802 slow virus carrier Sequence.

The immunoblotting of embodiment 3:Lenti801 and Lenti802 transient expression human serum albumin in people HEK293 is examined It surveys

1, the 1st day, 6 × 10 are planted in the cell culture well of the hole 12-⁵A HEK293 or C3A cell, cell culture fluid are 1mlDMEM (GIBCO)+10%FBS (GIBCO).Set 37 DEG C of carbon dioxide incubator cultures 12-16 hours.

2, the 2nd day, 1.5ml sterilizing EP pipe is taken, 2 μ g Lenti801 (or Lenti802) carrier DNAs are added thereto, add Enter the opti-MEM of 50 μ l, stands 5 minutes.Simultaneously in another 1.5ml sterilizing EP pipe, 50 μ lopti-MEM are added (GIBCO), 6 μ l lipofectamine Fugen HD (Roche) are added thereto, stand 5 minutes.

3, two pipe mixtures in step 2 are merged, softly mixes content with liquid-transfering gun, is stored at room temperature 20 minutes.

4, it is taken out from incubator by tissue culture plate, combined DNA and Fugen HD liposome solutions is equably added dropwise It into the cell in the hole 12-, slightly mixes, cell is put back into incubator and continues to cultivate.

5, the 3rd day, after 12-16 hours, turned with containing in the 1ml the being fully warmed-up fresh DMEM+10%FBS replacement hole 12- The culture medium of transfection reagent-DNA mixture, set continue in incubator culture be cultivated for 36 hours.

6, the 4th day, 48h after transfection, the original culture medium in culture hole, the serum-free being fully warmed-up with 1ml are removed RPMI1640 is washed cell 2 times, is added in 1ml serum-free DMEM to cell culture well, is put back into incubator and continues culture 24 Hour.

7, cell culture fluid 1000rpm is taken to be centrifuged 3min, transfer culture solution supernatant is set in 1.5ml pipe.Supernatant is in 4 DEG C of refrigerators It saves, use or minus 80 DEG C of refrigerator long-term preservations in 2 weeks.

8,20 μ l culture solution supernatants are taken, 2.5 μ l 5xSDS sample-loading buffers, 90 DEG C of heat denatured 3min are added.Sample is frequent After advising SDS-PAGE electrophoretic separation, human serum albumins is detected with conventional western blotting.

Embodiment 4:Lenti801 and Lenti802 starts marker gene expression in people HEK293

1, cell transfecting is completed referring to 3 step 1-5 of embodiment.With fluorescence microscope fluorescence protein gene whether table It reaches.

2, cell is taken out, cell culture fluid is absorbed.Volume cell in culture hole is washed with 1ml phosphate buffer.200 μ l are added 1xSDS sample-loading buffer lytic cell into cell culture well.

3, cell pyrolysis liquid is transferred in 1.5ml pipe, 12000rpm is centrifuged 15min, removal precipitating.25 μ l are taken to crack Liquid, 90 DEG C of 3min.On sample after SDS-PAGE electrophoresis, GenBank AAB30693 is detected with western blotting technology The expression of gene.

As a result as shown in figure 3, Lenti801 is in HK cells' HEK293 transient expression riddled basins human glutamine The testing result of synzyme.

Embodiment 5: transfection 293T cell prepares slow virus

For the slow virus packaging system used for Pax2 and VSV-G, 293T is incasing cells, culture medium be DMEM (GIBCO)+ 10%FBS (GIBCO).

1, the 1st day, 1.3 × 10 are planted in the cell culture well of the hole 6-⁶A 293T cell, cell culture fluid 2ml.Set 37 DEG C Carbon dioxide incubator culture 12-16 hours.

2, the 2nd day, 1.5ml sterilizing EP pipe is taken, 1.6 μ g Lenti802 (or Lenti801), 1.2 μ g Pax2,0.8 are added The opti-MEM of 100 μ l is added in μ gVSV-G carrier DNA, stands 5 minutes.In another 1.5ml sterilizing EP pipe, 100 μ are added 10.8 μ l lipofectamine Fugene HD (Roche) are added in lopti-MEM (GIBCO) thereto, stand 5 minutes.

3, two pipe mixtures in step 2 are merged, softly mixes content with liquid-transfering gun, is stored at room temperature 20 minutes.

4, it is taken out from incubator by tissue culture plate, DNA solution and Fugen HD liposome solutions is equably added drop-wise to It in the culture medium of 293T cell in the hole 6-, slightly mixes, cell is put back into cell incubator and continues to cultivate.

5, it after 12-16 hours, is replaced in the hole 6- with the 2ml culture medium being fully warmed-up containing transfection reagent-DNA mixture Culture medium, cell is put back in incubator continue culture be cultivated for 36 hours.

6, the 4th day, 48h after transfection, the supernatant containing virus is collected.By supernatant with 0.45 μm of filter be filled into sterilizing from In heart pipe.Virus is set 4 DEG C of refrigerators and is saved, and uses in 2 weeks.

Embodiment 6: slow-virus infection human hepatoma cell line HepG2/C3A establishes stable cell line.

1, the 1st day, the HepG2/C3A cell of cell to logarithmic growth phase is cultivated with the RPMI1640 containing 10%FBS, through pancreas After enzymic digestion counts, the every hole 24- inoculation 5 × 10⁴A cell, every 500 μ L of hole cell culture fluid, sets in cell incubator and cultivates 12-16 hours.

2, the 2nd day: taking out cell from incubator, 0.5 μ l8mg/mlPolybrene is added into cell culture fluid (Sigma), soft to mix.The 200 μ L of slow virus collected in embodiment 5 is added into cell again, mixes.It is put back into cell culture Continue to cultivate in case.

3, the 3rd day: replacing fresh medium after infection after 16-24 hours, the culture solution containing lentiviral particle is sucked out, again 500 μ LDMEM+10%FBS culture solutions are added to culture hole, continue to cultivate.

4, the 4th day: routinely cell passage operation amplifying cells.

As shown in figure 4, being C3A nucleus GFP positive cell after virus infection.As shown in figure 5, being through flow cytometer point Select later GFP positive stable cell strain.

Embodiment 7: the Coomassie brilliant blue detection of human serum albumins in stable cell line

1 inoculating cell 6x10⁵In RPM1640+10%FBS in the hole 12-, liquid is changed every other day, and culture to cell is covered with entirely Culture hole bottom.

2 removal culture mediums, are washed 2 times with the PBS for the 1ml being fully warmed-up.Then the 1ml of no any addition is added RPMI1640 culture medium sets cell incubator culture 24 hours.

3 take 20 μ l of supernatant, row SDS-PAGE to carry out coomassie brilliant blue staining；In addition 10 μ l is taken to carry out westernblot inspection It surveys.Fig. 6 is the coomassie brilliant blue staining testing result of the destination protein human serum albumin of stable cell line expression, it was confirmed that this hair Bright building by human serum albumins promoter drive in people's cell express human serum albumin gene carrier at The high efficient expression external source recombination human serum albumin in human liver cell of function, while the carrier overcomes human serum albumins and sieve Select the false positive cell clone frequency height marked divide in the carrier system with different promoters in screening and expression unbalanced Problem.

Embodiment 8: the inspection of human serum albumin gene promoter and human serum albumin CDS sequence in stable cell line genome It surveys

In human liver cell genomic DNA, human serum albumin Gene A LB is to be separated with multiple exons by multiple intrones The form opened exists, and only just has complete continuous ALB albumen coded sequence in the mRNA obtained after genetic transcription (CDS) exist.

It is characterized in that ALB gene opens by the genomic DNA of this patent embodiment 3-6 stable cell line obtained CDS (coded sequence), IRES sequence and the riddled basins of mover, the First Intron of ALB and whole ALB are to connect Continuous form is integrated in one or more positions in cellular genome.

Fig. 7 is for external source human serum albumin promoter in the genome of the stable cell line prepared by slow-virus infection and outside The inspection policies of source human serum albumin gene can detecte the presence of this feature nucleic acid fragment using the strategy.Using ALB base Because of the reverse primer of among the forward primer and albumin gene of the starting point of promoter or end, with HepG2/C3A The genomic DNA of Lenti802 cell is template, amplifiable to of corresponding size by archaeal dna polymerase chain reaction (PCR) DNA fragmentation, and DNA can be carried out using above-mentioned any one primer to the DNA fragmentation of PCR amplification and be routinely sequenced, the sequence of survey with Human serum albumin promoter and human serum albumin gene included in carrier Lenti802 are identical (or reverse complemental).

Specific implementation operation:

1, collect 1 × 10⁶A cell, routinely cellular genome extracting method extracts the genomic DNA of cell.

2, with primer 802GenomALBpro1F (SEQ ID NO.9) respectively with primer 802Genom1451-1421R (SEQ ID NO.10) and primer 802Genom2421R (SEQ ID NO.11) pairing, PCR amplification is carried out to the genomic DNA of extraction. Amplified production is separated through conventional DNA agarose gel electrophoresis, will respectively obtain 1.45kb (primer 802GenomALBpro1F and 802Genom1451-1421R) and the segment of 2.42kb (primer 802GenomALBpro1F and 802Genom2421R) product.

Pcr amplification reaction system (table 1):

Table 1

Genomic DNA template (is diluted to 50ng/ul)	1 μ l (50ng or so)
		Forward primer	0.75μl
Reverse primer	0.75μl
		dNTP	5 μ l (every kind of 200uM)
High-fidelity DNA polymerase (KOD archaeal dna polymerase)	1μl
		2 × KOD buffer	12.5μl
H2O	4μl
		Total volume	25μl

The program of PCR instrument amplification are as follows: referring to table 2

Table 2

Detection primer sequence used is as follows:

Table 3

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

Inspection policies:

Primer and human serum albumin 3 ' is held to hold primer using human serum albumin promoter 5 ', amplified production includes promoter 0.59kb and albumin gene 1.83kb is altogether 2.42kb.Using the end of human serum albumin promoter 5 ' and albumin middle position 3 ' end primers, amplified production include promoter and human serum albumin N-terminal coded sequence, total 1.45kb.

Testing result is as shown in figure 8, this is deposited in the stable cell line genome for detect slow-virus infection preparation by PCR In the testing result of external source human serum albumin promoter and external source human serum albumin gene.Swimming lane 1 (successively from left to right) is DNA Marker；

Swimming lane 2 is to be with 802GenomALBpro1F (SEQ ID NO.9) and 802Genom2421R (SEQ ID NO.11) Primer pair obtains the amplified production comprising promoter and albumin gene；

Swimming lane 3 is with 802GenomALBpro1F (SEQ ID NO.9) and 802Genom1451-1421R (SEQ ID It NO.10) is primer pair, the obtained amplified production comprising promoter and human serum albumin N-terminal coded sequence.

As it can be seen that having detected the presence of external source human serum albumin promoter and external source people's blood in the genome of stable cell line Albumin gene nucleotide fragments.

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Sequence table

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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

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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

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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

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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 55 60

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 Ile Ala 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 (17)

1. a kind of expression vector driven with albumin promoter, it is characterised in that: the expression vector includes expression cassette, described Expression cassette from 5 ' ends to 3 ' ends successively comprising albumin promoter and its First Intron, albumin gene, internal ribosome into Angle of striking sequence IRES, the second gene.

2. expression vector according to claim 1, it is characterised in that: the albumin promoter is that human serum albumins opens Mover.

3. expression vector according to claim 1, it is characterised in that: the albumin gene is selected from human serum albumins base Cause, dog serum albumin gene, cat Serum Albumin Gene, bovine serum albumin(BSA) gene, sheep blood serum albumin gene, Swine serum Albumin gene, horse serum albumin gene, mouse serum albumin gene, monkey serum albumin gene, albumin rabbit serum base Cause, it is preferable that the albumin gene is human serum albumin gene.

4. expression vector according to claim 1, it is characterised in that: second gene is riddled basins.

5. expression vector according to claim 4, it is characterised in that: the riddled basins are synthesized selected from glutamine It is enzyme gene, kalamycin resistance gene, tetracycline resistance gene, neomycin phosphotransferase gene, puromycin-resistant base, green Color fluorescence protein gene, red fluorescent protein gene.

6. expression vector according to claim 5, it is characterised in that: the riddled basins are human glutamine synthesis Enzyme gene or green fluorescence protein gene.

7. expression vector according to claim 1, it is characterised in that: the expression cassette also includes other controlling elements.

8. expression vector according to claim 1, it is characterised in that: the expression vector also includes skeleton.

9. expression vector according to claim 8, it is characterised in that: the skeleton be selected from retrovirus, slow virus with Adenoviral backbone, it is preferable that the skeleton is slow virus skeleton.

10. -9 described in any item expression vectors according to claim 1, it is characterised in that: the human serum albumins promoter And First Intron has nucleotide sequence or its variant sequence thereof shown in SEQ ID NO.1, the human serum albumins base Because having nucleotide sequence or its variant sequence thereof shown in SEQ ID NO.2, the internal ribosome entry site sequence IRES tool There are nucleotide sequence shown in SEQ ID NO.3 or its variant sequence thereof.

11. expression vector according to claim 5 or 6, it is characterised in that: GenBank AAB30693's gene has Nucleotide sequence shown in SEQ ID NO.4 or its variant sequence thereof, the green fluorescence protein gene have SEQ ID NO.5 institute The nucleotide sequence shown or its variant sequence thereof.

12. a kind of expression cell, it is characterised in that: the expression cell includes expression of any of claims 1-11 Carrier.

13. expression cell according to claim 12, it is characterised in that: it is thin that the cell is selected from people's cell, cat cell, dog Born of the same parents, ox cell, sheep cell, it is preferable that the cell is HK cells or human liver cell.

14. a kind of method for producing human serum albumins, it is characterised in that: the method includes allowing human serum albumins table Under conditions of reaching, expression cell described in claim 12 or 13, and the recycling human serum albumins are cultivated.

15. a kind of human serum albumins, it is characterised in that: the human serum albumins is described in any item by claim 1-11 Expression vector or the described in any item expression cell expression of claim 12-13, or prepared by method of claim 14 And it obtains.

16. application of the albumin promoter in driving albumin gene expression, which is characterized in that building starts comprising albumin The expression vector of son.

17. application according to claim 16, it is characterised in that: the albumin promoter is human serum albumins starting Son, the albumin are human serum albumins.