CN118813548B - Method and culture medium for expressing recombinant human albumin by using BTI-Tn-5B1-4 cells - Google Patents
Method and culture medium for expressing recombinant human albumin by using BTI-Tn-5B1-4 cells Download PDFInfo
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Abstract
本申请公开了一种利用BTI‑Tn‑5B1‑4细胞表达重组人白蛋白的方法及培养基，属于生物技术和基因技术领域。本申请方案中，针对BTI‑Tn‑5B1‑4细胞表达重组人白蛋白进行密码子优化，并且筛选表达效率较高的信号肽序列，并且通过对基础昆虫培养基进行优化，明显提高了利用BTI‑Tn‑5B1‑4细胞表达重组人白蛋白的效率。
The present application discloses a method and culture medium for expressing recombinant human albumin using BTI-Tn-5B1-4 cells, which belongs to the fields of biotechnology and gene technology. In the present application, codon optimization is performed for BTI-Tn-5B1-4 cells expressing recombinant human albumin, and signal peptide sequences with high expression efficiency are screened, and the efficiency of expressing recombinant human albumin using BTI-Tn-5B1-4 cells is significantly improved by optimizing the basic insect culture medium.
Description
Technical Field
The application relates to a method and a culture medium for expressing recombinant human albumin by utilizing BTI-Tn-5B1-4 cells, belonging to the field of biotechnology and gene technology.
Background
Human serum albumin (human serum albumin, HSA) is the most abundant protein in human blood plasma, accounting for 50-60% of the total protein in the blood plasma, HSA belongs to a single-chain non-glycosylated protein, has the composition of 585 amino acids, the molecular weight of 66.5kD, the isoelectric point of 4.7-4.9, 17 pairs of disulfide bonds and 1 free mercapto group in the molecule, and the whole molecule spirals into a heart. Human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules, etc. in body fluids while maintaining normal blood osmotic pressure.
Clinically, the human serum albumin can be used for treating shock and burn, supplementing blood loss caused by surgery, accident or massive hemorrhage, and can also be used as a plasma compatibilizer. Human serum albumin is mainly extracted from blood plasma, and because of limited sources of the blood plasma and the threat of blood-borne diseases such as AIDS, hepatitis and the like, people have great worry about the application of the serum albumin extracted from the blood plasma as medical application.
Recombinant protein drugs are protein drugs produced by applying genetic engineering techniques, and have become an important component of biological drugs in recent years. The production of recombinant protein medicine mainly includes four systems of prokaryotic protein expression, yeast protein expression, insect cell protein expression and mammal cell protein expression, and human serum albumin has been expressed and produced in colibacillus, yeast, animal cell and rice.
High Five (formally called BTI-Tn-5B 1-4) is an insect cell line, ovary cells derived from spodoptera frugiperda, found by BTI of the institute of plant in the united states, uses baculovirus or transfection, and expresses more recombinant proteins than lepidopteran insect cell lines such as Sf9 cells, which can be grown in the absence of serum, and can be cultured in a loosely attached state or suspension.
The insect cell expression protein has good expression level and relatively fast growth speed, the glycosylation modification of the protein is closer to that of a mammalian cell, baculovirus is amplified in an insect host cell, the generated recombinant protein is more similar to that of the protein expressed by the mammalian cell in post-transcriptional modification, the expressed product is closer to that of human serum albumin in molecular structure, physicochemical property and biological function, so that the expression product has strong biological activity, and compared with that of the albumin expressed by the mammalian cell, the expression product has lower culture medium cost and simpler growth requirement, is more suitable for industrial production, and has important significance in researching the expression of the human albumin by the insect cell, but no report of expressing the recombinant human albumin by using the BTI-Tn-5B1-4 cell is currently seen.
Disclosure of Invention
In order to solve the problems, the application provides a method and a culture medium for expressing recombinant human albumin by utilizing BTI-Tn-5B1-4 cells, and in the scheme of the application, codon optimization is carried out on the recombinant human albumin expressed by the BTI-Tn-5B1-4 cells, a signal peptide sequence with higher expression efficiency is screened, and the efficiency of expressing the recombinant human albumin by utilizing the BTI-Tn-5B1-4 cells is obviously improved by optimizing a basic insect culture medium.
According to one aspect of the present application, there is provided a Medium for culturing BTI-Tn-5B1-4 cells to express human albumin, the Medium being obtained by supplementing 10wt% FBS from Insect Medium Grace's instrument Medium, supplemented and adding ethanolamine and insulin, wherein the concentration of ethanolamine is 6 to 10. Mu.g/ml, and the concentration of insulin is 3 to 7. Mu.g/ml.
According to another aspect of the present application, there is provided a method for expressing recombinant human albumin using BTI-Tn-5B1-4 cells, the method comprising the step of culturing the BTI-Tn-5B1-4 cells to express recombinant human albumin after introducing the nucleotide sequence SEQ ID NO:5 encoding human albumin into the BTI-Tn-5B1-4 cells.
Optionally, the method comprises the steps of:
s1, carrying out passage on BTI-Tn-5B1-4 cells, and adding a culture medium to culture the cells to form a well-grown cell monolayer;
S2, preparing a recombinant vector by using the nucleotide sequence SEQ ID NO. 5 for encoding the human albumin and a vector pFastBacl;
S3, transforming the E.coli DH5 alpha competent cells by using the recombinant vector, and then transforming the obtained recombinant plasmid into E.coli DH10Bac competent cells to obtain recombinant baculovirus plasmid Bacmid;
S4, transfecting the recombinant baculovirus plasmid Bacmid into BTI-Tn-5B1-4 cells, and performing suspension culture.
Optionally, the suspension culture in step S4 uses the above-mentioned culture medium.
According to another aspect of the present application there is provided a free nucleotide encoding human albumin, the nucleotide having the sequence shown in SEQ ID NO. 5.
According to another aspect of the present application there is provided an expression vector comprising the above-described free nucleotide encoding human albumin.
Optionally, the carrier is pFastBacl.
According to another aspect of the present application there is provided a recombinant BTI-Tn-5B1-4 cell comprising the above-described free nucleotide encoding human albumin or the above-described expression vector.
According to another aspect of the present application, there is provided the use of the above-described method for expressing recombinant human albumin using BTI-Tn-5B1-4 cells in the preparation of recombinant human albumin.
The beneficial effects of the application include, but are not limited to:
1. According to the method for expressing recombinant human albumin by utilizing BTI-Tn-5B1-4 cells, disclosed by the application, the codon optimization is carried out on the BTI-Tn-5B1-4 cells, and the signal peptide is screened, so that the secretion effect of the human albumin in the BTI-Tn-5B1-4 cells can be obviously improved by the HBM signal peptide, and the efficiency of expressing the recombinant human albumin by utilizing the BTI-Tn-5B1-4 cells is obviously improved by 166% compared with that of an unoptimized sequence.
2. According to the method for expressing recombinant human albumin by using BTI-Tn-5B1-4 cells, disclosed by the application, the method is optimized for a culture medium, the early growth state of the BTI-Tn-5B1-4 cells can be obviously improved by adding ethanolamine and insulin, and compared with a TNM-FH complete culture medium conventionally used, the method has better human albumin expression efficiency and is improved by 73%.
3. According to the method for expressing the recombinant human albumin by using the BTI-Tn-5B1-4 cells, the efficient expression of the recombinant human albumin in the BTI-Tn-5B1-4 cells is successfully realized, the molecular structure, the physicochemical property and the biological function of the recombinant human albumin are closer to those of human serum albumin, and meanwhile, compared with the expression of the albumin by using mammalian cells, the method has lower culture medium cost and simpler growth requirement, is suitable for industrial production, and has important industrial application prospect and economic value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
FIG. 1 is a schematic diagram of pFastBacl vectors according to example 2 of the present application;
FIG. 2 is a diagram showing SDS-PAGE results according to example 3 of the present application;
FIG. 3 is a Western-blot result chart relating to example 3 of the present application;
FIG. 4 is a graph showing the results of the expression level of rHSA according to example 4 of the present application;
FIG. 5 is a graph showing the results of the expression level of rHSA according to example 5 of the present application;
FIG. 6 is a graph showing the results of the expression level of rHSA according to example 6 of the present application.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples, and the raw materials and catalysts in the examples of the present application are commercially available unless otherwise specified. The various media, reagents, E.coli, cell line reagents, tool enzymes, etc. used in the examples and test examples are commercially available. The procedures used in the examples and test cases are not specifically described, and are conventional in the art, for example, refer to the molecular cloning laboratory Manual (Sambrook J & Russell DW. Molecular cloning: a laboratory Manual 2001), or the instructions provided by the manufacturer of the product.
Cells and reagents or kits are BTI-Tn-5B1-4 cells (Gibco Co.), E.coli DH 5. Alpha. (full gold Biotechnology Co., ltd.), E.coli DH10Bac (Bomeid. Gene technology Co., ltd.), plasmid pFastBacI (Invitrogen Co., ltd.), culture medium Sf-900 II SFM (Gibco Co.), insect cell transfection kit (Gibco Co.), recombinant plasmid pFastBacI-technical services such as sequencing and primer synthesis (BamHI and XholI) provided by Shanghai Bioengineering Co., ltd.), ethanolamine (Shanghai Bioengineering Co., ltd.), sodium butyrate (Shanghai Bioengineering Co., ltd.), insulin (Shanghai Bioengineering Co., ltd.).
The following describes the inventive solution by means of specific examples.
Example 1 optimization of human serum albumin genes
Gene expression is regulated and affected by a variety of factors, such as codon usage bias, ribosome binding, mRNA structure, etc., whereas mRNA sequences encoding the same protein sequence are possible, and higher expression results may be achieved by optimizing the codons of the sequence to match the expression profile of the highly expressed gene in the target host.
In the scheme of the application, the mature peptide amino acid sequence of the adopted human serum albumin rHSA is shown as SEQ ID NO. 1, and in addition, the mature peptide amino acid sequence also comprises an HBM signal peptide sequence consisting of 21 amino acids at the N end, the amino acid sequence is shown as SEQ ID NO. 2, and the mature peptide nucleotide sequence rHSA-S0 of the human serum albumin rHSA before optimization is shown as SEQ ID NO. 3.
The experimenter optimizes the mature peptide coding sequence of rHSA according to the codon preference of BTI-Tn-5B1-4, and obtains 3 optimized nucleotide sequences rHSA-S1, rHSA-S2 and rHSA-S3 by considering signal peptide factors, the gene sequences of the optimized nucleotide sequences are shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 in sequence, and the optimized sequences are subjected to chemical total synthesis by Shanghai biological engineering Co-Ltd and are verified by sequencing of the Co-Ltd. The base composition of the optimized gene is shown in Table 1.
Table 1 rHSA base composition of optimized Gene sequence
EXAMPLE 2 construction of expression vectors
The nucleotide sequences shown in SEQ ID NO 3-6 are artificially synthesized, and the nucleotide sequences are respectively inserted into pFastBacl vectors in a seamless cloning mode, a model diagram of pFastBacl vectors is shown in figure 1, gene sequence synthesis and vector construction services are completed by Shanghai biological engineering Inc., and constructed correct plasmids are named as pFastBacl-rHSA-S0, pFastBacl-rHSA-S1, pFastBacl-rHSA-S2 and pFastBacl-rHSA-S3 respectively.
The carrier construction process comprises the following steps:
the nucleotide sequence shown as SEQ ID NO. 3-6 is artificially synthesized by Shanghai biological engineering Co., ltd, bamHI (GGATCC) enzyme cutting site and HBM signal peptide nucleotide sequence SEQ ID NO. 7 are respectively introduced at the 5 'end of the synthesized sequence, and XhoI (CTCGAG) enzyme cutting site is introduced at the 3' end.
The target gene and pFastBacI vector obtained by double digestion with BamHI and XhoI were used, respectively, and the double digestion system was as shown in Table 2 below.
TABLE 2 double enzyme digestion System
The reaction conditions were 37 ℃ for 4h. After the completion of the reaction, agarose gel electrophoresis was performed, and then the target band was directly recovered using TIANGEN DNA recovery kit. The recovered empty vector and the double cleavage product of the target gene were ligated with T4 DNA ligase (molar ratio of target gene: vector DNA=3:1), and the ligation reaction system was as shown in Table 3 below.
TABLE 3 ligation reaction System
The ligation was carried out overnight at 16 ℃. 10 μl of the above-mentioned ligation product was used to transform E.coli DH 5. Alpha. Competent cells, which were plated on LB solid plates (tryptone 10g, agar powder 15g, yeast extract 5g, naCl 10g, deionized water to 1L) with ampicillin resistance overnight, white clone colonies were picked up on the plates the following day and subjected to gene sequencing identification, recombinant plasmids pFastBacl-rHSA-S0, pFastBacl-rHSA-S1, pFastBacl-rHSA-S2 and pFastBacl-rHSA-S3 with correct sequencing were transferred into E.coli DH10Bac competent cells, which were plated for cultivation, and white spots were selected from the cultured white spot colonies to extract the baculovirus plasmid Bacmid.
Example 3 expression and characterization of human serum albumin
The identified baculovirus plasmid Bacmid was transferred into BTI-Tn-5B1-4 cells using a transfection kit.
The specific transfection comprises the following steps:
1) BTI-Tn-5B1-4 cells were seeded in six well cell culture plates (1 mL per well) at a cell density of 2X 10 6 cells/mL and incubated for 2h;
2) Diluting L-2 mug recombinant baculovirus plasmid Bacmid with 100 mu L of Sf-900 II SFM, diluting a transfection reagent Cellfectin with 100 mu L of Sf-900 II SFM, mixing the two dilutions, incubating for 40min at room temperature, and adding 1mL of Sf-900 II SFM to prepare a transfection mixture;
3) Removing the cell culture supernatant in the step 1), adding the transfection mixture in the step 2), incubating for 4-6 hours at 28 ℃, discarding the transfection mixture, and adding 2mL of Sf-900 II SFM, and culturing for 3-4 days in a 28 ℃ constant temperature incubator;
4) Collecting cell culture supernatant, inoculating P1 generation cells into new BTI-Tn-5B1-4 cells according to 2% volume, continuously culturing for two generations so as to amplify virus virulence, and observing and recording P3 generation cell morphology change when P3 generation cells are obtained, and carrying out expression identification on the P3 generation cells, wherein the identification method adopts SDS-PAGE identification and Western Blot verification.
SDS-PAGE and Western Blot identification, in which samples were subjected to 12% SDS-PAGE (260V, 40 min) and stained with Coomassie brilliant blue gel for 1.5 hours, and the images were scanned and observed after pure water decolorization, SDS-PAGE results are shown in FIG. 2, and it can be seen from FIG. 2 that a band appears at about 66.5kDa, whose position is consistent with that expected, initially indicating correct expression of recombinant human serum albumin, SDS-PAGE electrophoresis samples were transferred to PVDF membrane for Western Blot identification, and Western Blot results are shown in FIG. 3, and it was found that the expression of recombinant human serum albumin in BTI-Tn-5B1-4 cells was successful at about 66.5 kDa.
EXAMPLE 4 results of human serum albumin production and expression level
BTI-Tn-5B1-4 cells were inoculated into a 5L flask, subjected to suspension culture at 27℃until the initial cell mass was 3 to 6X 10 5 cells/mL, 1L of TNM-FH complete Medium (prepared by adding 10wt% FBS to Insect Medium Grace 'S select Medium, supplemented, wherein Grace' S select Medium, supplemented and FBS were all purchased from Invitrogen) was added, and subjected to suspension culture at 120rpm, the next day of P3 generation virus was added at a ratio of 1:50, and the supernatant was collected until the seventh day of culture, and the expression level of rHSA was measured by HPLC, and the control group was the unoptimized rHSA-S0 sequence, and the measurement results of the expression level were shown in FIG. 4.
As can be seen from the results of FIG. 4, rHSA was successfully expressed in BTI-Tn-5B1-4 cells after codon-optimized HSA sequence and addition of the selected HBM signal peptide. rHSA can be expressed in BTI-Tn-5B1-4 cells after binding to a signal peptide if the sequence is not optimized, but the expression amount is small. Therefore, in the scheme of the application, the signal peptide sequence with the sequence shown as SEQ ID NO. 2 can enable rHSA to be successfully expressed in insect BTI-Tn-5B1-4 cells, and after codon optimization, the protein expression quantity of rHSA-S2 (SEQ ID NO. 5) is obviously improved, and compared with the non-optimized scheme, the expression quantity of rHSA is improved by about 166 percent.
Example 5 screening and optimization of Signal peptides
The experimenter also performed design and screening work of signal peptides based on BTI-Tn-5B1-4 cells, with alternative signal peptide sequences shown in Table 4 below.
TABLE 4 Signal peptide sequence numbering and sequence information therefor
The nucleotide sequences of the different signal peptides in Table 4 are respectively matched with SEQ ID NO. 5, baculovirus plasmid Bacmid is prepared by adopting the same scheme in the scheme, and the expression and detection of recombinant human albumin are carried out by adopting the same conditions as in example 4, and the detection result is shown in FIG. 5.
As can be seen from the results of FIG. 5, the HBM signal peptide has the best effect on the expression level of rHSA when it is coordinated with the mature peptide amino acid sequence SEQ ID NO:1 of human serum albumin rHSA, compared with other signal peptides, wherein the HBM signal peptide amino acid sequence SEQ ID NO:2 (corresponding nucleotide sequence SEQ ID NO: 7) has the best effect on secretion and expression of rHSA in BTI-Tn-5B1-4 cells, compared with the HSA signal peptide, GP64 signal peptide, chi signal peptide and HIV-ENV signal peptide.
In the scheme of the application, the baculovirus plasmid Bacmid obtained after the recombinant plasmid is composed of the nucleotide sequences SEQ ID NO 5 and pFastBacl is used for producing rHSA after the BTI-Tn-5B1-4 cells are infected, so that the expression level of the recombinant human albumin is the highest, and compared with the expression level of the rHSA in the non-optimized scheme, the expression level of the recombinant human albumin is improved by about 166%, and obvious technical progress is obtained.
Example 6 suspension Medium optimization
When the experimenter carried out the culture of BTI-Tn-5B1-4 cells to express recombinant human albumin, TNM-FH complete Medium prepared by supplementing 10wt% FBS with Insect Medium Grace's select Medium, supplemented (Grace's select Medium, supplemented and FBS are all purchased from Invitrogen) was used, however, the experimenter found that the early growth rate of BTI-Tn-5B1-4 cells during the culture was slow, resulting in lower expression efficiency during the post-expression of recombinant human albumin, and for this reason the experimenter tried to optimize the composition of the Medium, it was said that other culture conditions were the same as those of example 4 except that the composition of the Medium specifically described in Table 5 was different, and the recombinant human albumin was detected by the same HPLC method as in example 4, and the expression amount detection result was shown in FIG. 6.
TABLE 5 optimization of Medium composition
According to the results shown in FIG. 6, the TNM-FH complete medium is cultivated and optimized, and meanwhile, ethanolamine and insulin are added, so that a better synergistic process can be achieved, the expression quantity of recombinant human albumin is obviously improved, compared with the expression quantity of rHSA which is not optimized, the expression quantity of rHSA is improved by about 73%, and experimental staff find that the BTI-Tn-5B1-4 cells adopting the optimized medium TNM-FH-3 enter the logarithmic growth phase earlier and have higher cell density by detecting the growth curve conditions of the TNM-FH complete medium and the optimized medium TNM-FH-3 early BTI-Tn-5B1-4 cells, so that the expression efficiency of the recombinant human albumin is obviously improved.
The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (2)
1. A method for expressing recombinant human albumin by using BTI-Tn-5B1-4 cells is characterized by comprising the steps of introducing a nucleotide sequence SEQ ID NO 5 encoding human albumin into BTI-Tn-5B1-4 cells and culturing the BTI-Tn-5B1-4 cells to express recombinant human albumin;
the method specifically comprises the following steps:
s1, carrying out passage on BTI-Tn-5B1-4 cells, and adding a culture medium to culture the cells to form a well-grown cell monolayer;
S2, preparing a recombinant vector by utilizing the nucleotide sequence SEQ ID NO.5 for encoding the human albumin and a vector pFastBacl, wherein the recombinant vector comprises an HBM signal peptide, the amino acid sequence of the HBM signal peptide is shown as SEQ ID NO. 2, the nucleotide sequence of the HBM signal peptide is shown as SEQ ID NO. 7, and the nucleotide sequence SEQ ID NO. 7 of the HBM signal peptide is positioned at the N end of the nucleotide sequence SEQ ID NO.5 for encoding the human albumin;
S3, transforming the E.coli DH5 alpha competent cells by using the recombinant vector, and then transforming the obtained recombinant plasmid into E.coli DH10Bac competent cells to obtain recombinant baculovirus plasmid Bacmid;
S4, transfecting the recombinant baculovirus plasmid Bacmid into BTI-Tn-5B1-4 cells, and performing suspension culture;
The Medium for suspension culture was obtained by supplementing 10wt% FBS from Insect Medium Grace's instrument Medium, supplemented with ethanolamine at an addition concentration of 8. Mu.g/ml and insulin at an addition concentration of 5. Mu.g/ml.
2. The application of the recombinant BTI-Tn-5B1-4 cells in the production of human albumin is characterized in that the recombinant BTI-Tn-5B1-4 cells comprise a recombinant vector prepared by utilizing a nucleotide sequence SEQ ID NO. 5 for encoding human albumin and a vector pFastBacl, the recombinant vector comprises an HBM signal peptide, the amino acid sequence of the HBM signal peptide is shown as SEQ ID NO. 2, the nucleotide sequence of the HBM signal peptide is shown as SEQ ID NO. 7, and the nucleotide sequence SEQ ID NO. 7 of the HBM signal peptide is positioned at the N end of the nucleotide sequence SEQ ID NO. 5 for encoding human albumin.
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