CN113637675A - Production method of human serum albumin, nucleotide sequence, expression vector and expression system - Google Patents
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Abstract
The invention belongs to the field of production of human serum albumin, and particularly relates to a production method, a nucleotide sequence, an expression vector and an expression system of human serum albumin. The nucleotide sequence is shown in SEQ ID NO. 1. The nucleotide sequence of the invention optimizes the codon of the human serum albumin gene according to the codon preference of CHO cells, and can improve the basic expression quantity of target genes. In addition, the invention further adopts serum-free medium-low temperature culture, and small molecular additives are added to efficiently express the human serum albumin in a CHO cell line.
Description
Technical Field
The invention belongs to the field of production of human serum albumin, and particularly relates to a production method, a nucleotide sequence, an expression vector and an expression system of human serum albumin.
Background
Human Serum Albumin (HSA) is a protein in Human plasma. Human serum albumin can transport fatty acids, cholesterol, amino acids, steroid hormones, metal ions and many therapeutic molecules etc. in body fluids: while maintaining normal osmotic pressure of the blood. In clinic, human serum albumin can be used for treating shock and burn, supplementing blood loss caused by operation, accident or hemorrhage, and can also be used as blood plasma bulking agent. Human serum albumin is mainly extracted from blood plasma, and due to the limited source of blood plasma and the threat of blood-borne diseases such as AIDS and hepatitis, people have great concerns about the application of the serum albumin extracted from blood plasma as a medicine.
Recombinant protein drugs are protein drugs produced by applying genetic engineering techniques, and in recent years, recombinant protein drugs have become important components of biological drugs. The production of recombinant protein drugs mainly comprises four major systems: prokaryotic protein expression, yeast protein expression, insect cell protein expression and mammalian cell protein expression. At present, albumin is expressed and produced in escherichia coli, yeast, animal cells and rice to produce human serum albumin. Compared with other expression systems, the recombinant protein produced by mammalian cells can be correctly folded, assembled and post-translationally modified, and has the advantage of being closer to the molecular structure of human protein, so the mammalian cells become the main expression host of protein drugs. Currently, approximately 70% of the approved recombinant protein drugs on the market are derived from Chinese hamster ovary Cells (CHO). However, the expression level of HSA produced by CHO is still low, the time required for obtaining high-expression engineering cell strains is long, the cost of large-scale cell culture is high, and the like, so that the cost of producing protein drugs by mammalian cells is high. There is a need for improving mammalian cell expression systems, further increasing the expression level of recombinant proteins, enhancing the stability of cell lines, reducing production costs, and ensuring product quality and safety.
For example, Chinese patent application with publication No. CN106220726A discloses a method for constructing recombinant human serum albumin and an expression vector thereof, and the expression level of HSA reaches 150-180 mg/L after the expression vector is optimized. The key link of CHO cell production recombinant protein is except expression vector, and downstream cell culture and process have industrial production significance.
Disclosure of Invention
The invention aims to provide a nucleotide sequence which can realize the high-efficiency expression of an HSA target gene in CHO cells.
The second objective of the invention is to provide an expression vector of the nucleotide sequence.
The third object of the present invention is to provide an expression system comprising the above expression vector.
The fourth purpose of the invention is to provide a method for improving the expression of the recombinant human serum albumin of the CHO cells.
In order to achieve the above purpose, the technical scheme of the nucleotide sequence of the invention is as follows:
a nucleotide sequence is shown as SEQ ID NO. 1.
The nucleotide sequence of the invention optimizes the codon of the human serum albumin gene according to the codon preference of CHO cells, and can improve the basic expression quantity of target genes.
The technical scheme of the expression vector of the invention is as follows:
an expression vector comprising the nucleotide sequence described above.
The expression vector can be constructed by inserting the gene sequence shown in SEQ ID NO.1 into the multiple cloning sites of the starting vector by the conventional method in the field. Specifically, an HSA gene sequence shown in SEQ ID NO.1 is artificially synthesized, the HSA gene sequence is connected to a starting vector by using a cloning technology, and a recombinant expression vector is obtained by sequence identification and screening.
Preferably, the starting vector for constructing the expression vector is pWTY9.3.
The technical scheme of the expression system of the invention is as follows:
an expression system comprising the above expression vector, obtained by transfecting CHO cells with the expression vector.
Preferably, the CHO cell is one or more of CHO-S, CHO-K1 and CHO-DG 44.
The vector containing pWTY9.3-HSA is transfected into CHO-S cell strain, and a stable CHO-HSA cell pool is obtained through culture and screening. The stable CHO-HSA cell pool is capable of high expression of HSA.
The technical scheme of the production method of the human serum albumin is as follows:
a method for producing human serum albumin comprises the following steps:
(1) constructing an expression vector containing a nucleotide sequence shown in SEQ ID NO. 1;
(2) transfecting an expression vector to a CHO cell strain, and culturing and screening to obtain a stable CHO-HSA cell pool;
(3) the stable CHO-HSA cell pool is cultured in suspension and serum-free.
The production method of the human serum albumin of the invention constructs a stable CHO-HSA cell pool by constructing an expression vector containing the HSA coding gene, thereby realizing the high-efficiency expression of the HSA target gene in CHO cells.
The CHO-HSA cell pool is cultured by expanding the cell strain from 96-well plate to 24-well plate and then to 6-well plate, and suspension serum-free culture is adopted. Preferably, in step (3), the stable CHO-HSA cell pool is cultured at 37 ℃ to logarithmic phase and then transferred to 33 ℃ for low temperature culture.
Preferably, in the step (3), sodium butyrate and hydrocinnamic acid are added during the suspension serum-free culture. Further preferably, the final concentrations of the sodium butyrate and the hydrocinnamic acid added are respectively 1.0-3.0 mol/L and 0.2-1.0 mol/L.
The conventional CHO cell culture medium can be selected, and preferably, in the step (3), the culture medium used for the suspension serum-free culture is CHO Pro Grow
And (3) serum-free culture medium.
Experiments prove that HSA can be successfully expressed in CHO cells, and the expression level of recombinant HSA protein in the CHO cells can be remarkably improved based on low-temperature and small-molecule additives under the same conditions.
The method for producing recombinant human serum albumin by mammalian cells has an O-glycosylation post-translational modification mode completely consistent with human cells, thereby overcoming the defect that the prior human serum albumin produced by yeast and rice and the albumin from human cells have incomplete consistent post-translational modification modes, and improving the functions of the human serum albumin.
Drawings
FIG. 1 is a schematic diagram of an expression vector pWTY9.3 used in the examples of the present invention;
FIG. 2 shows the Western blot results of HSA expression in CHO cells according to the present invention;
FIG. 3 is a graph of cell density of HSA of the present invention under different temperature conditions;
FIG. 4 is a graph showing the cell activity of HSA of the present invention under various temperature regulations;
FIG. 5 is a graph showing HSA expression levels under different temperature regulations in the present invention;
FIG. 6 is a graph of the cell density of HSA of the present invention under the action of various additives;
FIG. 7 is a graph showing the cell activity of HSA of the present invention under the effect of various additives;
FIG. 8 is a graph showing the expression levels of HSA in the present invention under the effect of various additives.
Detailed Description
The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; various media, reagents, E.coli JM109, cell-based reagents, and instrument enzymes used in the examples and test examples were commercially available. The pWTY9.3 plasmid is a product of Henan Punuoyi biological products research institute, Inc. The procedures in the examples and experimental examples are not specifically indicated, but are generally performed by conventional techniques in the art, for example, by referring to molecular cloning, a laboratory manual, compiled by Sambrook et al (Sambrook J & Russell DW. molecular cloning: a laboratory Manual.2001), or by instructions provided by manufacturers of products.
Nucleotide sequence examples
Example 1HSA Gene sequence
According to the literature report on the HSA amino acid sequence (Genbank no: CAA 01491); according to the codon preference of CHO cells, codon optimization is carried out, the Codon Adaptation Index (CAI) of the optimized HSA gene sequence is 0.97, the GC content is 0.59, the expression quantity is improved by 1.5 times compared with that before optimization, and the sequence is shown as SEQ ID NO. 1. Introducing a signal peptide sequence shown as SEQ ID NO. 2.
The HSA gene sequence before optimization is shown in SEQ ID NO. 3.
The amino acid sequence of the recombinant human serum albumin is shown in SEQ ID NO. 4.
Second, example of expression vector
Example 2 construction of recombinant expression vector containing HSA Gene
This example provides a method for constructing a recombinant expression vector comprising an HSA-encoding gene, comprising the steps of:
artificially synthesizing SEQ ID NO.1, introducing HindIII (AAGCTT) and NheI (GCTAGC) enzyme cutting sites at the 5 'end and the 3' end of a synthetic sequence respectively, adding a Kozak sequence at the downstream of the HindIII enzyme cutting site, and synthesizing by a general biological system (Anhui) limited company.
The synthetic HSA sequences were double-digested with HindIII/NheI, respectively, while the pWTY9.3 plasmid DNA vector was double-digested with HindIII/NheI (see FIG. 1 for vector structure). And identifying the digestion result by agarose gel electrophoresis, and recovering the HSA sequence fragment and pWTY9.3 linear plasmid DNA after digestion by gel.
The double-enzyme system of HSA sequence is: HSA sequence 10. mu.L (1. mu.g/. mu.L), 10 XNEBuffer 2.1Buffer 3.0. mu.L, HindIII/NheI (10U/. mu.L) each 1.0. mu.L, make up water to 30. mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h.
The double enzyme cutting system of the pWTY9.3 plasmid is as follows: pIRES-Neo plasmid 5. mu.L (1. mu.g/. mu.L), 10 XNEBuffer 2.1Buffer 2.0. mu.L, HindIII/NheI (10U/. mu.L) each 0.5. mu.L, make up water to 20. mu.L; the enzyme digestion conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h.
The digested HSA sequence fragment and pWTY9.3 linear plasmid DNA (molar ratio 5:1) were ligated at 25 ℃ for 5min using a ligation kit of NEB TM. The ligation product was added to a competent cell suspension of Escherichia coli (E.coli) JM109 strain to transform, 150. mu.L of the transformant was inoculated onto an LB plate containing ampicillin, cultured overnight at 37 ℃ and single colony was selected for subculture. Extracting recombinant plasmids, carrying out double enzyme digestion (HindIII/NheI) verification, taking the plasmids with correct enzyme digestion verification for sequencing verification, and respectively naming the constructed correct plasmids as pWTY9.3-HSA.
Third, example of expression System
Example 3 cell transfection and Stable cell pool selection
The construction process of the expression system of the embodiment is as follows:
(1) CHO cells were cultured in complete medium containing 10% fetal bovine serum, and when the cells were in logarithmic growth phase, the collected cells were seeded into 24-well plates at 15 ten thousand/ml. The next day when the cells were growing fast, 1. mu.l of the lipofectin was used
pWTY9.3-HSA containing 1. mu.g of the vector was transfected.
(2) The vector-transfected CHO cells were subjected to pressure selection using DMEM/F12 medium containing blasticidin (15. mu.g/ml). After the untransfected control cells were completely killed with blasticidin, the surviving cells in the experimental group were stably transfected cell lines. Reducing the concentration of blasticidin in the culture medium to 10 μ g/ml for subculture expansion, wherein the cells are cultured at 37 deg.C and 5% CO2Cultured in an incubator for 20 passages to obtain CHO cell pools stably transfected with HSA, and analyzed by Western blot for HSA expression, and the results are shown in FIG. 2. As can be seen from the results in fig. 2, HSA was successfully expressed in stable CHO cell pools.
The CHO cells used in this example were CHO-S cells. In other embodiments, CHO-K1 and CHO-DG44 cell lines can be used to achieve the corresponding improvements.
Fourth, example of production method of human serum albumin
Example 4
The method for producing human serum albumin of this example comprises the following steps:
the CHO cell pool (stable CHO-HSA cell pool) of example 3 stably transfected with HSA was transferred to a 125mL suspension flask with an initial cell size of 5-6X 106one/mL, 30mL of CHO Pro Grow was added
Serum-free medium, suspension culture at 120rpm, cell density analysis with a serum cell counter and cell viability analysis with trypan blue staining daily.
This example is a culture at 37 ℃ at normal temperature; and collecting cell supernatants until the seventh day of culture, and detecting the expression level of HSA in each group by ELISA.
Example 5
The method for producing human serum albumin of this example is essentially the same as described in example 4, except that the stable CHO-HSA cell pool is first cultured at 37 ℃ to logarithmic phase, and then transferred to a low temperature of 33 ℃. Specifically, the culture was carried out at 37 ℃ at normal temperature until the fourth day, and then the culture was carried out at 33 ℃.
Example 6
The method for producing human serum albumin of this example was substantially the same as the method described in example 4, except that the culture conditions were low-temperature culture at 33 ℃.
Examples 7 to 11
The culture and analysis of the stable CHO-HSA cell pool were carried out according to the culture method of example 5, and sodium butyrate and hydrocinnamic acid were added respectively the next day of cell culture, with the final concentrations of the additions of each example as shown in Table 1 below. Cell density was analyzed daily using a serum cell counter and cell viability was analyzed using trypan blue staining. And collecting cell supernatants until the seventh day of culture, and detecting the expression level of HSA in each group by ELISA.
TABLE 1 addition of sodium butyrate and hydrocinnamic acid in examples 7 to 11
Fifth, example of experiment
This experiment example tested the differences in cell density, cell activity and HSA expression levels of stable CHO-HSA cell pools under different culture conditions.
(1) Different culture temperatures
The results of comparing the cell density, cell activity and HSA volume expression level in the methods of examples 4 to 6 are shown in FIGS. 3 to 5.
As is clear from fig. 3 to 5, the cell density of the method of example 4 was slightly high, and the cell activities of the groups were not significantly different. The volume expression levels of HSA in the three groups of examples 4, 5 and 6 were 190.15mg/L, 304.46mg/L and 213.55mg/L, respectively, with the highest expression level in example 5 followed by example 6.
(2) Whether to add small molecule additives
The results of comparing the cell density, cell activity and HSA volume expression level in the methods of examples 7 to 11 are shown in FIGS. 6 to 8.
As is clear from fig. 6 to 8, the cell density and the cell activity of each group of cells were not significantly different. In examples 7 to 11, the HSA was expressed in the volume at 311.05mg/L, 359.46mg/L, 436.34mg/L, 517.49mg/L, 412.06mg/L and 303.14mg/L, respectively, and the expression level was highest in the fourth group (example 10) and the third group (example 9) in the following group, as compared with the control group.
<110> Xinxiang medical college of biologicals institute of Henan Punuo Yi
<120> production method of human serum albumin, nucleotide sequence, expression vector and expression system
<160> 4
<170> PatentIn version 3.5
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Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
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Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
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Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
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Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
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Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
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Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
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Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
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Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
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Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
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Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
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Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
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Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
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Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
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Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
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Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
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Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
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Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
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Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
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Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
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Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
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Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
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Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
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Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
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Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
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Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
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Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
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Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
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Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
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Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585
Claims (10)
1. A nucleotide sequence is shown as SEQ ID NO. 1.
2. An expression vector comprising the nucleotide sequence of claim 1.
3. The expression vector according to claim 2, wherein the starting vector for constructing the expression vector is pWTY9.3.
4. An expression system comprising the expression vector of claim 2, wherein the expression vector is obtained by transfecting CHO cells.
5. The expression system of claim 4, wherein the CHO cell is one or more of CHO-S, CHO-K1, CHO-DG 44.
6. A method for producing human serum albumin, comprising the steps of:
(1) constructing an expression vector containing a nucleotide sequence shown in SEQ ID NO. 1;
(2) transfecting an expression vector to a CHO cell strain, and culturing and screening to obtain a stable CHO-HSA cell pool;
(3) the stable CHO-HSA cell pool is cultured in suspension and serum-free.
7. The method of claim 6, wherein in step (3), the stable CHO-HSA cell pool is cultured at 37 ℃ to logarithmic phase and then transferred to a low temperature of 33 ℃.
8. The method for producing human serum albumin according to claim 6 or 7, wherein sodium butyrate and hydrocinnamic acid are added in step (3) during the suspension serum-free culture.
9. The method for producing human serum albumin according to claim 8, wherein the sodium butyrate and the hydrocinnamic acid are added in the final concentrations of 1.0 to 3.0mol/L and 0.2 to 1.0mol/L, respectively, in step (3).
10. Process for the preparation of human serum albumin according to claim 6 or 7The production method is characterized in that in the step (3), the culture medium used for suspension serum-free culture is CHO Pro
And (3) serum-free culture medium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265964A1 (en) * | 2003-04-25 | 2004-12-30 | Martin Allen | Inducers of recombinant protein expression |
| CN106220726A (en) * | 2016-05-25 | 2016-12-14 | 西北民族大学 | Recombinant human serum albumin and the construction method of expression vector thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265964A1 (en) * | 2003-04-25 | 2004-12-30 | Martin Allen | Inducers of recombinant protein expression |
| CN106220726A (en) * | 2016-05-25 | 2016-12-14 | 西北民族大学 | Recombinant human serum albumin and the construction method of expression vector thereof |
Non-Patent Citations (1)
| Title |
|---|
| ZHOU JIANG等: "Sodium Butyrate Stimulates Monoclonal Antibody Over-Expression in CHO Cells by Improving Gene Accessibility.", 《BIOTECHNOL. BIOENG》 * |
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