A method and culture medium for expressing recombinant human albumin using BTI-Tn-5B1-4 cells

Abstract

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

[0001] The present application relates to a method and culture medium for expressing recombinant human albumin using BTI-Tn-5B1-4 cells, and belongs to the field of biotechnology and gene technology. Background Art

[0002] Human serum albumin (HSA) is the most abundant protein in human plasma, accounting for 50-60% of total plasma protein. HSA is a single-chain non-glycosylated protein composed of 585 amino acids, with a molecular weight of 66.5kD and an isoelectric point between 4.7 and 4.9. The molecule contains 17 pairs of disulfide bonds and 1 free thiol group, and the entire molecule is coiled into a heart shape. In body fluids, human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules, while maintaining normal blood osmotic pressure.

[0003] In clinical practice, human serum albumin can be used to treat shock and burns, to supplement blood loss caused by surgery, accidents or massive bleeding, and can also be used as a plasma expander. Human serum albumin is mainly extracted from plasma. Due to the limited source of plasma and the threat of blood-borne diseases such as AIDS and hepatitis, people have great concerns about the use of serum albumin extracted from plasma for medical purposes.

[0004] Recombinant protein drugs are protein drugs produced by genetic engineering technology. In recent years, recombinant protein drugs have become an important part of biological drugs. The production of recombinant protein drugs mainly includes four major systems: prokaryotic protein expression, yeast protein expression, insect cell protein expression and mammalian cell protein expression. At present, some people have expressed and produced human serum albumin in Escherichia coli, yeast, animal cells and rice.

[0005] High Five (officially known as BTI-Tn-5B1-4) is an insect cell line that originated from the ovarian cells of the cabbage looper, discovered by the American plant research institute BTI. High Five cells use baculovirus or transfection and express more recombinant proteins than lepidopteran insect cell lines such as Sf9 cells. They can grow in the absence of serum and can be cultured in a loosely attached state or in suspension.

[0006] Insect cell expressed proteins have good expression levels and relatively fast growth rates, and the glycosylation modification of the protein is closer to that of mammalian cells. Baculovirus will be amplified in insect host cells, and the recombinant protein produced is more similar to the protein expressed by mammalian cells in terms of post-transcriptional modification. The expressed product is closer to human serum albumin in terms of molecular structure, physicochemical properties and biological function. Therefore, the expressed product has strong biological activity. Compared with mammalian cell expressed albumin, it has lower culture medium cost and simpler growth requirements, and is more suitable for industrial production. Therefore, it is of great significance to study the expression of human albumin by insect cells. However, there is no report on the expression of recombinant human albumin using BTI-Tn-5B1-4 cells. Summary of the invention

[0007] In order to solve the above problems, a method and culture medium for expressing recombinant human albumin using BTI-Tn-5B1-4 cells are provided. In the present application, codon optimization is performed on BTI-Tn-5B1-4 cells expressing recombinant human albumin, and signal peptide sequences with higher expression efficiency are screened. In addition, the efficiency of expressing recombinant human albumin using BTI-Tn-5B1-4 cells is significantly improved by optimizing the basic insect culture medium.

[0008] According to one aspect of the present application, a culture medium for culturing BTI-Tn-5B1-4 cells to express human albumin is provided, wherein the culture medium is obtained by adding 10wt% FBS to Grace's Insect Medium, Supplemented and adding ethanolamine and insulin, wherein the ethanolamine is added at a concentration of 6 to 10 μg / ml, and the insulin is added at a concentration of 3 to 7 μg / ml.

[0009] According to another aspect of the present application, a method for expressing recombinant human albumin using BTI-Tn-5B1-4 cells is provided, the method comprising the steps of introducing the nucleotide sequence SEQ ID NO: 5 encoding human albumin into BTI-Tn-5B1-4 cells, and then culturing the BTI-Tn-5B1-4 cells to express recombinant human albumin.

[0010] Optionally, the method comprises the following steps:

[0011] S1, subculturing BTI-Tn-5B1-4 cells, adding culture medium and culturing the cells to form a well-growing cell monolayer;

[0012] S2, using the nucleotide sequence encoding human albumin SEQ ID NO: 5 and vector pFastBacl to prepare a recombinant vector;

[0013] S3, the recombinant vector is transformed into E. coli DH5α competent cells, and then the obtained recombinant plasmid is transformed into E. coli DH10Bac competent cells to obtain the recombinant bacmid;

[0014] S4. Transfect BTI-Tn-5B1-4 cells with the recombinant bacmid plasmid and culture them in suspension.

[0015] Optionally, the suspension culture in step S4 uses the above-mentioned culture medium.

[0016] According to another aspect of the present application, a free nucleotide encoding human albumin is provided, and the sequence of the nucleotide is shown in SEQ ID NO:5.

[0017] According to another aspect of the present application, an expression vector containing the above-mentioned free nucleotide encoding human albumin is provided.

[0018] Optionally, the vector is pFastBacl.

[0019] According to another aspect of the present application, a recombinant BTI-Tn-5B1-4 cell containing the above-mentioned free nucleotide encoding human albumin or the above-mentioned expression vector is provided.

[0020] According to another aspect of the present application, there is provided the use of the method for expressing recombinant human albumin using the above-mentioned BTI-Tn-5B1-4 cells in the preparation of recombinant human serum albumin.

[0021] The beneficial effects of this application include but are not limited to:

[0022] 1. According to the method of expressing recombinant human albumin using BTI-Tn-5B1-4 cells of the present application, by codon optimization for BTI-Tn-5B1-4 cells and screening for signal peptides, it is found that HBM signal peptide can significantly improve the secretion effect of human albumin in BTI-Tn-5B1-4 cells, thereby significantly improving the efficiency of expressing recombinant human albumin using BTI-Tn-5B1-4 cells, which is as high as 166% compared with the unoptimized sequence.

[0023] 2. According to the method of expressing recombinant human albumin using BTI-Tn-5B1-4 cells in the present application, the culture medium is optimized, and the early growth state of BTI-Tn-5B1-4 cells can be significantly improved by adding ethanolamine and insulin. Compared with the conventionally used TNM-FH complete culture medium, the human albumin expression efficiency is better, which is increased by 73%.

[0024] 3. According to the method of expressing recombinant human albumin using BTI-Tn-5B1-4 cells of the present application, the efficient expression of recombinant human albumin in BTI-Tn-5B1-4 cells is successfully achieved. While being closer to human serum albumin in terms of molecular structure, physicochemical properties and biological functions, it also has lower culture medium costs and simpler growth requirements than mammalian cell expression of albumin, is suitable for industrial production, and has important industrial application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0026] Figure 1 This is a schematic diagram of the pFastBacl vector involved in Example 2 of the present application;

[0027] Figure 2 This is the SDS-PAGE result diagram involved in Example 3 of the present application;

[0028] Figure 3 This is the Western-blot result diagram involved in Example 3 of the present application;

[0029] Figure 4 This is a graph showing the rHSA expression results involved in Example 4 of the present application;

[0030] Figure 5 This is a graph showing the rHSA expression results involved in Example 5 of the present application;

[0031] Figure 6 This is a graph showing the rHSA expression results involved in Example 6 of the present application. DETAILED DESCRIPTION

[0032] The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are purchased through commercial channels. All kinds of culture media, reagents, Escherichia coli, cell line reagents, tool enzymes, etc. used in the examples and test examples are commercially available. If not specifically specified, the relevant operations in the examples and test examples are all conventional technical means in the field, such as with reference to the molecular cloning laboratory manual (Sambrook J & Russell DW.Molecular cloning:a laboratory manual.2001) compiled by Sambrook, etc., or the instructions provided by the product manufacturer, etc.

[0033] Cells and reagents or kits: BTI-Tn-5B1-4 cells (Gibco); E. coli DH5α (Quanshijin Biotechnology Co., Ltd.); E. coli DH10Bac (Bomed Gene Technology Co., Ltd.); plasmid pFastBacI (Invitrogen), culture medium Sf-900 II SFM (Gibco); insect cell transfection kit (Gibco); recombinant plasmid pFastBacI-gene sequencing and primer synthesis (BamHI and XholI) and other technical services were provided by Shanghai Sangon Biotechnology Co., Ltd.; ethanolamine (Shanghai Sangon Biotechnology Co., Ltd.); sodium butyrate (Shanghai Sangon Biotechnology Co., Ltd.); insulin (Shanghai Sangon Biotechnology Co., Ltd.).

[0034] The present application scheme is described below through specific embodiments.

[0035] Example 1 Optimization of human serum albumin gene

[0036] Gene expression is regulated and influenced by many factors, such as codon usage preference, ribosome binding, mRNA structure, etc. There are multiple possibilities for mRNA sequences encoding the same protein sequence. By optimizing the codons of the sequence to match the expression profile of highly expressed genes in the target host, the resulting gene sequence may achieve a higher expression effect.

[0037] In the present application, the amino acid sequence of the mature peptide of human serum albumin rHSA used is shown in SEQ ID NO: 1. In addition, it also contains an HBM signal peptide sequence composed of 21 amino acids at the N-terminus, the amino acid sequence of which is shown in SEQ ID NO: 2. The nucleotide sequence of the mature peptide of human serum albumin rHSA before optimization, rHSA-S0, is shown in SEQ ID NO: 3.

[0038] The experimenters optimized the mature peptide coding sequence of rHSA according to the codon preference of BTI-Tn-5B1-4 cells, and obtained three optimized nucleotide sequences rHSA-S1, rHSA-S2 and rHSA-S3 after considering the signal peptide factor, and their gene sequences are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The optimized sequences were chemically synthesized by Shanghai Shenggong Biotechnology Co., Ltd. and sequenced and verified by the company. The base composition of the optimized gene is shown in Table 1.

[0039] Table 1 Base composition of rHSA optimized gene sequence

[0040]

[0041] Example 2 Construction of expression vector

[0042] The nucleotide sequences shown in SEQ ID NO: 3 to 6 were artificially synthesized and inserted into the pFastBacl vector by seamless cloning. The schematic diagram of the pFastBacl vector is shown in FIG. Figure 1 As shown, gene sequence synthesis and vector construction services were completed by Shanghai Sangon Biotechnology Co., Ltd., and the correctly constructed plasmids were named pFastBacl-rHSA-S0, pFastBacl-rHSA-S1, pFastBacl-rHSA-S2 and pFastBacl-rHSA-S3, respectively.

[0043] The vector construction process includes the following steps:

[0044] Shanghai Sangon Biotechnology Co., Ltd. was commissioned to artificially synthesize the nucleotide sequences shown in SEQ ID NOs: 3 to 6, and a BamHI (GGATCC) restriction site and an HBM signal peptide nucleotide sequence SEQ ID NO: 7 were introduced at the 5' end of the synthetic sequence, and an XhoI (CTCGAG) restriction site was introduced at the 3' end.

[0045] The target gene and pFastBacI vector were double-digested with BamHI and XhoI, respectively. The double-digestion system is shown in Table 2 below.

[0046] Table 2 Double enzyme digestion system

[0047]

[0048] The reaction conditions were 37°C for 4 hours. After the reaction, agarose gel electrophoresis was performed, and then the target band was directly recovered using the TianGen DNA recovery kit. The recovered empty vector and the double enzyme digestion product of the target gene were connected with T4 DNA ligase (the molecular molar ratio was target gene: vector DNA = 3:1). The connection reaction system is shown in Table 3 below.

[0049] Table 3 Ligation reaction system

[0050]

[0051] Connect overnight at 16°C. Take 10 μl of the above-mentioned connection product to transform E. coli DH5α competent cells, spread on LB solid plates containing ampicillin resistance (tryptone 10g, agar powder 15g, yeast extract 5g, NaCl 10g, deionized water to 1L) and culture overnight; the next day, pick white clone colonies on the plate and perform gene sequencing identification, and transfer the correctly sequenced recombinant plasmids pFastBacl-rHSA-S0, pFastBacl-rHSA-S1, pFastBacl-rHSA-S2 and pFastBacl-rHSA-S3 into E. coli DH10Bac competent cells, spread on plates for culture; select white spots from the cultured blue-white colonies and extract their bacmid plasmids.

[0052] Example 3 Expression and identification of human serum albumin

[0053] The identified bacmid was transformed into BTI-Tn-5B1-4 cells using a transfection kit.

[0054] The specific transfection steps include the following:

[0055] 1) BTI-Tn-5B1-4 cells were cultured at 2×10 6 cells / mL, inoculated into six-well cell culture plates (1 mL per well) and incubated for 2 h;

[0056] 2) Take 1~2μg of recombinant bacmid plasmid and dilute it with 100μL of Sf-900 II SFM; take transfection reagent Cellfectin and dilute it in 100μL of Sf-900 II SFM; mix the two dilutions obtained above, incubate at room temperature for 40min, and then add 1mL of Sf-900 II SFM to prepare the transfection mixture;

[0057] 3) Remove the cell culture supernatant in step 1) and add the transfection mixture in step 2) and incubate at 28°C for 4-6 hours; discard the transfection mixture, add 2 mL of Sf-900 II SFM, and culture in a 28°C constant temperature incubator for 3-4 days;

[0058] 4) Collect the cell culture supernatant and inoculate it into new BTI-Tn-5B1-4 cells at 2% volume as P1 cells, and culture them for two consecutive generations to amplify the virulence of the virus; when it comes to P3 cells, observe and record the morphological changes of P3 cells, and perform expression identification on P3 cells by SDS-PAGE identification and Western Blot verification.

[0059] SDS-PAGE and Western Blot identification: The samples were subjected to 12% SDS-PAGE electrophoresis (260V, 40min), and the gel was stained with Coomassie brilliant blue for 1.5 hours. After decolorization with pure water, the electrophoresis image was scanned and observed; the SDS-PAGE results are shown in Figure 2 As shown, from Figure 2 It can be seen that a band appears at about 66.5 kDa, and its position is consistent with the expectation, which preliminarily indicates that the recombinant human serum albumin is correctly expressed; the SDS-PAGE electrophoresis sample is transferred to a PVDF membrane for Western Blot identification. The Western Blot results are shown in Figure 3 As shown, a target band was found at about 66.5 kDa, indicating that recombinant human serum albumin was successfully expressed in BTI-Tn-5B1-4 cells.

[0060] Example 4 Human serum albumin scale-up production and expression results

[0061] BTI-Tn-5B1-4 cells were inoculated into 5L culture flasks and cultured in suspension at 27°C. The initial cell count was 3-6×10 5 1L TNM-FH complete medium (Grace's Insect Medium, Supplemented with 10wt% FBS, both Grace's Insect Medium, Supplemented and FBS purchased from Invitrogen) was added and suspended at 120rpm. On the second day, P3 virus was added at a ratio of 1:50. The cell supernatant was collected on the seventh day of culture and the expression of rHSA was detected by HPLC. The control group was the unoptimized rHSA-S0 sequence. The expression detection results are shown in the figure. Figure 4 shown.

[0062] Depend on Figure 4 The results show that after the codon-optimized HSA sequence and the addition of the selected HBM signal peptide, rHSA can be successfully expressed in BTI-Tn-5B1-4 cells. If the sequence is not optimized, rHSA can be expressed in BTI-Tn-5B1-4 cells after combining with the signal peptide, but the expression amount is very low. Therefore, in the present application, the signal peptide sequence shown in SEQ ID NO: 2 is selected to enable rHSA to be successfully expressed in insect BTI-Tn-5B1-4 cells, and after codon optimization, the protein expression level of rHSA-S2 (SEQ ID NO: 5) is significantly improved, and the expression level of rHSA in the unoptimized solution is increased by about 166%.

[0063] Example 5 Screening and optimization of signal peptides

[0064] The experimenters also designed and screened signal peptides based on BTI-Tn-5B1-4 cells, and the candidate signal peptide sequences are shown in Table 4 below.

[0065] Table 4 Signal peptide sequence numbers and sequence information

[0066]

[0067] The nucleotide sequences of the different signal peptides in Table 4 were matched with SEQ ID NO: 5 respectively, and the bacmid plasmid was prepared by the same scheme in this scheme, and the expression and detection of recombinant human albumin were carried out under the same conditions as in Example 4. The detection results are as follows: Figure 5 shown.

[0068] according to Figure 5 The results show that compared with other signal peptides, the HBM signal peptide amino acid sequence SEQ ID NO: 2 (corresponding nucleotide sequence SEQ ID NO: 7), combined with the mature peptide amino acid sequence SEQ ID NO: 1 of human serum albumin rHSA, has the best rHSA expression effect. It can be seen that the HBM signal peptide is more conducive to the secretion and expression of rHSA in BTI-Tn-5B1-4 cells than the HSA signal peptide, GP64 signal peptide, Chi signal peptide and HIV-ENV signal peptide.

[0069] In the present application, the bacmid obtained by combining the nucleotide sequence SEQ ID NO: 5 with pFastBacl to form a recombinant plasmid is then used to infect BTI-Tn-5B1-4 cells for the production of rHSA, which has the highest expression level of recombinant human albumin. Compared with the unoptimized solution, the expression level of rHSA is increased by about 166%, achieving significant technical progress.

[0070] Example 6 Optimization of suspension culture medium

[0071] When the experimenters cultured BTI-Tn-5B1-4 cells to express recombinant human albumin, they used TNM-FH complete medium, which was composed of insect medium Grace's Insect Medium, Supplemented supplemented with 10wt% FBS (Grace's Insect Medium, Supplemented and FBS were purchased from Invitrogen). However, the experimenters found that the early growth rate of BTI-Tn-5B1-4 cells was slow during the culture process, resulting in low expression efficiency in the later expression of recombinant human albumin. For this reason, the experimenters tried to optimize the composition of the culture medium. It should be noted that except for the special culture medium components recorded in Table 5, the other culture conditions were the same as those in Example 4, and the recombinant human albumin was detected using the same HPLC method as in Example 4. The expression detection results are shown in FIG. Figure 6 shown.

[0072] Table 5 Optimization of culture medium components

[0073]

[0074] according to Figure 6 The results showed that by culturing and optimizing the TNM-FH complete medium, the addition of ethanolamine and insulin can achieve a good synergistic enhancement process, significantly increasing the expression of recombinant human albumin. The expression of rHSA was increased by about 73% compared with the unoptimized scheme. In addition, by detecting the growth curves of early BTI-Tn-5B1-4 cells in the TNM-FH complete medium and the optimized medium TNM-FH-3, the experimenters found that the BTI-Tn-5B1-4 cells using the optimized medium TNM-FH-3 entered the logarithmic growth phase earlier and had a higher cell density, thus significantly improving the expression efficiency of recombinant human albumin.

[0075] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for expressing recombinant human albumin using BTI-Tn-5B1-4 cells, characterized in that: The method comprises the steps of introducing the nucleotide sequence SEQ ID NO: 5 encoding human albumin into BTI-Tn-5B1-4 cells, and then culturing the BTI-Tn-5B1-4 cells to express recombinant human albumin; The specific steps include: S1, subculturing BTI-Tn-5B1-4 cells, adding culture medium and culturing the cells to form a well-growing cell monolayer; S2. Prepare a recombinant vector using the nucleotide sequence SEQ ID NO: 5 encoding human albumin and a vector pFastBacl, wherein the recombinant vector comprises an HBM signal peptide, wherein the amino acid sequence of the HBM signal peptide is as shown in SEQ ID NO: 2, and the nucleotide sequence of the HBM signal peptide is as shown in SEQ ID NO: 7, wherein the nucleotide sequence of the HBM signal peptide SEQ ID NO: 7 is located at the N-terminus of the nucleotide sequence SEQ ID NO: 5 encoding human albumin; S3, the recombinant vector is transformed into E. coli DH5α competent cells, and then the obtained recombinant plasmid is transformed into E. coli DH10Bac competent cells to obtain the recombinant bacmid; S4, transfecting the recombinant bacmid plasmid into BTI-Tn-5B1-4 cells for suspension culture; The suspension culture medium is obtained by adding 10 wt % FBS to Grace's Insect Medium, Supplemented and adding ethanolamine and insulin. The concentration of ethanolamine added is 8 μg / ml, and the concentration of insulin added is 5 μg / ml.

2. Use of a recombinant BTI-Tn-5B1-4 cell in the production of human albumin, characterized in that: The recombinant BTI-Tn-5B1-4 cells include a recombinant vector prepared using the nucleotide sequence SEQ ID NO: 5 encoding human albumin and the vector pFastBacl, the recombinant vector includes an HBM signal peptide, the amino acid sequence of the HBM signal peptide is shown in SEQ ID NO: 2, the nucleotide sequence of the HBM signal peptide is shown in SEQ ID NO: 7, and the nucleotide sequence of the HBM signal peptide SEQ ID NO: 7 is located at the N-terminus of the nucleotide sequence SEQ ID NO: 5 encoding human albumin.

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