A method for producing human serum albumin using insect cells, nucleotide sequence and expression vector

Abstract

The present invention relates to a method, nucleotide sequence and expression vector for producing human serum albumin using insect cells, and belongs to the field of the production of human serum albumin. The nucleotide sequence of the human serum albumin of the present invention is shown as SEQ ID NO.1. According to the codon preference of insect cells, the present invention optimizes the codons of the human serum albumin gene and combines it with a signal peptide sequence shown as SEQ ID NO.2, so as to achieve the expression of human serum albumin in Sf9 cells. The present invention further discloses a method for producing human serum albumin using insect cells. By adding small molecule additives during the production process, the expression level of human serum albumin in Sf9 cells can be effectively increased.

Description

Technical Field

[0001] The present invention relates to a method, nucleotide sequence and expression vector for producing human serum albumin using insect cells, and belongs to the field of human serum albumin production. Background Art

[0002] Human Serum Albumin (HSA) is a single-chain non-glycosylated protein composed of 585 amino acids with a molecular weight of 66KDa. Under normal circumstances, hepatocytes synthesize a certain amount of serum albumin every day to maintain the dynamic balance of the total HSA amount in the human body. HSA not only plays a role in maintaining plasma osmotic pressure and promoting wound healing, but also serves as a carrier substance in the blood and participates in the transportation of various biomolecules such as hormones, metal ions, and drugs in the blood. Human serum albumin extracted from human plasma is mainly used clinically to treat hypoproteinemia, acute gastrointestinal bleeding and other diseases caused by blood loss, plastic surgery, etc., and has good application prospects. However, the traditional method of extracting HSA from blood is often restricted by various factors and is inevitably infected by various pathogenic microorganisms such as HIV, HBV, HCV, etc., which has become a prominent issue of international concern.

[0003] In recent years, with the continuous development of genetic engineering technology, recombinant protein drugs have become an important part of biopharmaceuticals. Currently, in the production of recombinant protein drugs, four protein expression systems are mainly applied: prokaryotic protein expression system, yeast protein expression system, insect cell protein expression system, and mammalian cell expression system. Compared with prokaryotic and yeast expression systems, the insect cell expression system is similar to mammalian cells in terms of post-translational modifications of proteins such as glycosylation and phosphorylation; compared with the mammalian cell expression system, the production conditions of insect cells are simple, without the need for CO2, and the temperature only needs to be 27°C. In addition, the insect cell expression system also has the advantages of large cloning capacity, high protein expression level, easy screening, and high biosafety, so it is widely used in many aspects such as biopharmaceutical production and basic research. According to incomplete statistics, more than 1,000 foreign genes have been successfully expressed in the insect cell expression system, and about 95% of the foreign recombinant proteins have biological activities similar to those of natural proteins.

[0004] At present, people have successfully expressed human serum albumin using prokaryotic expression systems (such as Escherichia coli and Bacillus subtilis), yeast expression systems, rice, tobacco, and mammalian cell (CHO, COS, etc.) expression systems, but there has been no successful expression in the insect cell expression system. Summary of the Invention

[0005] To solve the above problems, the object of the present invention is to provide a nucleotide sequence encoding human serum albumin, which can achieve the high-efficiency expression of the HSA target gene in insect cells.

[0006] The second object of the present invention is to provide an expression vector containing the above nucleotide sequence encoding human serum albumin.

[0007] The third object of the present invention is to provide a method for producing human serum albumin using insect cells.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A nucleotide sequence encoding human serum albumin, wherein the nucleotide sequence is as shown in SEQ ID NO.1.

[0010] The nucleotide sequence encoding human serum albumin of the present invention optimizes the codons of the human serum albumin gene according to the codon preference of insect cells, which can improve the basal expression level of the target gene.

[0011] An expression vector containing the above nucleotide sequence encoding human serum albumin.

[0012] The expression vector containing the above nucleotide sequence encoding human serum albumin of the present invention refers to a viral expression vector carrying the nucleotide sequence of human serum albumin of the present invention. Specifically, the original vector of the expression vector can be sp1-Bac.

[0013] The above expression vector can introduce the nucleotide sequence shown in SEQ ID NO:1 into the original vector by conventional methods in the art.

[0014] Preferably, a signal peptide sequence is added to the 5' end of the nucleotide sequence encoding human serum albumin in the expression vector; the nucleotide sequence of the signal peptide is as shown in SEQ ID NO.2.

[0015] By adding a signal peptide sequence as shown in SEQ ID NO.2 to the 5' end of the nucleotide sequence encoding human serum albumin in the expression vector, the signal peptide cooperates with the codon-optimized nucleotide sequence encoding human serum albumin, and can successfully express human serum albumin in insect cells.

[0016] A method for producing human serum albumin using insect cells, which is characterized in that the above nucleotide sequence is introduced into insect cells to obtain a recombinant virus containing the human serum albumin sequence, and the virus is collected and used to infect insect cells to express human serum albumin.

[0017] The method for producing human serum albumin using insect cells according to the present invention has the expressed HSA protein with natural post-translational modifications, overcoming the drawbacks that when expressing HSA in bacteria, insoluble polymers are produced and need to be reprocessed to form mature soluble proteins; it also overcomes the deficiencies that when expressing in yeast, the yield is very low and a considerable part of HSA has been cleaved, as well as the limitations such as high production costs and complex processes when expressing in mammalian cells. While ensuring the function of human serum albumin, it has the advantages of cost savings, high yield, and large-scale preparation.

[0018] Preferably, the method for producing human serum albumin using insect cells includes the following steps:

[0019] (1) Construct a donor vector containing the nucleotide sequence shown in SEQ ID NO.1;

[0020] (2) Transform the donor vector into DH10Bac competent cells to obtain recombinant bacmids;

[0021] (3) Transfect the recombinant bacmid into insect cells to obtain a recombinant virus containing the nucleotide sequence of human serum albumin;

[0022] (4) Infect insect cells with the recombinant virus and perform suspension serum-free culture.

[0023] The method for producing human serum albumin using insect cells according to the present invention realizes the efficient expression of the HSA target gene in insect cells by constructing a donor vector containing the HSA coding gene, obtaining a recombinant bacmid containing the HSA coding gene, transfecting insect cells to obtain a recombinant virus containing the nucleotide sequence of human serum albumin, and infecting and culturing insect cells with the recombinant virus.

[0024] Further preferably, in step (1), the donor vector contains a signal peptide sequence added to the 5' end of the target gene; the nucleotide sequence of the signal peptide is as shown in SEQ ID NO.2.

[0025] The signal peptide with the nucleotide sequence shown in SEQ ID NO.2 obtained through preliminary screening has a high adaptability with the codon-optimized HSA nucleotide sequence, which can effectively increase the expression level of HSA in insect cells. At the same time, this signal peptide is beneficial for insect cells to express and secrete HSA into the supernatant medium, and is more suitable for large-scale industrial production requirements.

[0026] Further preferably, in step (4), sodium butyrate and decitabine are added during the suspension serum-free culture.

[0027] Further preferably, the final concentrations of sodium butyrate and decitabine added in step (4) are 0.5 - 10 mmol / L and 5 - 50 nmol / L, respectively.

[0028] Experiments have proved that HSA can be successfully expressed in insect cells, and adding the combination of sodium butyrate and decitabine at the above concentrations can significantly improve the expression level of recombinant HSA protein in insect cells.

[0029] Further preferably, when performing the suspension serum-free culture in step (4), the medium used is InsectPro serum-free medium.

[0030] Preferably, the insect cells are one or more of Sf9, Sf21, and High Five.

[0031] Sf9, Sf21, and High Five cells can be adherent or suspension-cultured, and have the characteristics of low production cost, high cell density, and high protein expression level, providing a good host for the high-efficiency expression of HSA. Brief Description of the Drawings

[0032] Figure 1 It is a schematic diagram of the donor vector sp1-Bac used in Experimental Example 2 of the present invention;

[0033] Figure 2 It is the Western blot result of HSA expressed by Sf9 cells in Experimental Example 4 of the present invention;

[0034] Figure 3 It is the cell density diagram of HSA under the action of different concentration additive combinations in Experimental Examples 5-8 of the present invention;

[0035] Figure 4 It is the cell viability diagram of HSA under the action of different concentration additive combinations in Experimental Examples 5-8 of the present invention;

[0036] Figure 5 It is the HSA expression level diagram of HSA under the action of different concentrations of additive combinations in Experimental Examples 5-8 of the present invention. Detailed Embodiments

[0037] The present invention will be further described below in conjunction with the detailed embodiments, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0038] The situations of some biological materials, experimental reagents, experimental equipment, etc. involved in the following examples and experimental examples are briefly introduced as follows:

[0039] Example 1 of a Nucleotide Sequence Encoding Human Serum Albumin

[0040] In this example, the nucleotide sequence encoding human serum albumin is shown as SEQ ID NO.1.

[0041] Example 1 of an expression vector containing a nucleotide sequence encoding human serum albumin

[0042] The expression vector in this example contains a nucleotide sequence encoding human serum albumin. The nucleotide sequence of human serum albumin is shown as SEQ ID NO.1, and the original vector of the expression vector is sp1 - Bac.

[0043] Example 1 of a method for producing human serum albumin using insect cells

[0044] In this example, the sequence shown as SEQ ID NO.1 is introduced into insect cells to obtain recombinant insect cells expressing human serum albumin. The recombinant insect cells are cultured to express human serum albumin. A signal peptide sequence is added to the 5' end of the sequence shown as SEQ ID NO.1; the nucleotide sequence of the signal peptide is shown as SEQ ID NO.2.

[0045] The insect cells are Sf9 cells. In other implementation cases, using Sf21 or High Five cell lines can also achieve the same effect as Sf9 cells.

[0046] Experimental Example 1 Nucleotide sequence encoding human serum albumin

[0047] The HSA gene sequence (GenBank: AF542069.1) was retrieved from Genbank and optimized according to the codon preference of insect cells. The GC content of the optimized HSA gene is 54.92%, and the codon adaptation index (CAI) is 0.92. Its sequence is shown as SEQ ID NO.1. The optimized sequence was chemically synthesized in its entirety by General Biology (Anhui) Co., Ltd. and verified by sequencing by this company. The HSA nucleotide sequence before optimization is shown as SEQ ID NO.4.

[0048] The amino acid sequence of recombinant human serum albumin is shown as SEQ ID NO.3.

[0049] Experimental Example 2 Construction of HSA recombinant vector and generation of HSA virus bacmid

[0050] This experimental example provides a method for constructing a recombinant vector containing the HSA coding gene, including the following steps:

[0051] 2.1 Construction of the donor vector

[0052] Synthesize the nucleotide sequence shown in SEQ ID NO.1 and insert it downstream of the signal peptide in the sp1-Bac vector by seamless cloning. The nucleotide sequence of the signal peptide is as shown in SEQ ID NO.2, and the schematic diagram of the sp1-Bac vector is as Figure 1 shown. The gene sequence synthesis and vector construction services were completed by Bio (Anhui) Co., Ltd., and the correctly constructed plasmid was named sp1-Bac-HSA.

[0053] 2.2 Generation of HSA virus bacmid

[0054] Transform the extracted sp1-Bac-HSA donor vector (1 μg) into DH10Bac competent cells, then add 600 μL of LB medium and resuscitate for 1 hour. Take 10 μL of the transformed bacterial liquid and inoculate it onto a triple-antibiotic LB plate containing tetracycline, gentamicin, and kanamycin, and culture overnight at 37 °C. Pick a single colony for subculture, and extract the HSA bacmid, amplify it with Puc / M13 primers, and verify by sequencing.

[0055] Experimental Example 3 Cell Transfection and Acquisition of Recombinant Virus

[0056] This experimental example includes cell transfection and HSA virus packaging to obtain recombinant virus, and the steps are as follows:

[0057] Culture Sf9 cells at 27 °C in a complete medium containing 10% fetal bovine serum (Grace insect medium + 10% serum + 1% double antibody). When the cell density reaches 90%, collect the cells and inoculate them into a 24-well plate at 150,000 / ml. Perform cell transfection the next day, and transfect 6 μl of liposome transfection reagent with 3 μg of HSA bacmid.

[0058] After 72 hours of transfection, centrifuge to collect the supernatant and verify by Western blot. At this time, the packaged virus is the P1 generation. Subsequently, inoculate Sf9 cells into a 24-well plate at 150,000 / ml. The next day, infect with the P1 generation virus at a ratio of 1:20. Observe the signs of infection after 24 - 48 hours, and collect the supernatant after 72 - 96 hours. At this time, the packaged virus is the P2 generation; obtain the P3 virus in the same way, measure the virus titer with a kit, and store it frozen in the dark.

[0059] Experimental Example 4 Production of Human Serum Albumin

[0060] Inoculate Sf9 cells into a 125 mL culture flask and culture them in suspension at 27 °C. The initial cell amount is 3 - 6×10 5per mL, add 30 ml of Insect Pro Grow serum-free medium, and culture with suspension at 120 rpm. On the second day, add the P3-generation virus at a ratio of 1:50. Analyze the cell density with a serum cell counter every day, and analyze the cell viability by trypan blue staining. Collect the cell supernatant on the seventh day of culture, and detect the expression level of HSA by Western blot. The control groups are the original HSA sequence without optimization and the HSA sequence without the addition of a signal peptide, which are subjected to virus packaging and protein expression as described above, and the cell supernatants are collected.

[0061] The results of Western blot detection are as Figure 2 shown. It can be seen from the figure that after adding the selected signal peptide to the codon-optimized HSA sequence, HSA can be successfully expressed in Sf9 cells. If the sequence is not optimized, HSA can be expressed in Sf9 cells after binding with the signal peptide, but the expression level is very low; if the sequence is not optimized and no signal peptide is added, HSA cannot be successfully expressed in Sf9 cells. This fully shows that selecting the signal peptide sequence shown in SEQ ID NO.2 can enable HSA to be successfully expressed in insect cells, and after codon optimization, the protein expression level is significantly increased.

[0062] Experimental Examples 5-8 Effects of Small Molecule Additives on the Yield of Human Serum Albumin

[0063] According to the culture method of Experimental Example 4, culture and analyze Sf9 cells. Add sodium butyrate and decitabine on the first day of cell culture. The final addition amounts of each experimental example are shown in Table 1 below. Analyze the cell density with a serum cell counter every day, and analyze the cell viability by trypan blue staining. Collect the cell supernatant on the seventh day of culture, and detect the expression level of HSA in each group by ELISA.

[0064] Table 1 Usage Concentrations of Sodium Butyrate and Decitabine in Experimental Examples 5-8

[0065]

[0066] Among them, during the culture process, test the differences in cell density, cell viability, and HSA expression level of Sf9 cells with different combinations of small molecule additives at different concentrations.

[0067] Compare the cell density, cell viability, and HSA volume expression level (a common detection method for protein yield, reflecting the cell production capacity, with units such as mg / L or g / L, etc., which can truly reflect the content of the target protein in the cell culture supernatant and is closer to the detection in industrial production) of the culture methods in Experimental Examples 5-8. The results are as Figures 2 to 5 shown.

[0068] From Figures 3 to 4It can be seen that the combined use of sodium butyrate and decitabine can effectively reduce the viable cell density and cell viability in each group.

[0069] It can be seen from Figure 5 that the volume expression levels of HSA in the four groups in Experimental Examples 5-8 are 196.08 mg / L, 127.97 mg / L, 123.84 mg / L, and 148.61 mg / L respectively. Compared with the control group (99.90 mg / L), the amount of HSA is significantly increased. When the concentration of sodium butyrate in the culture medium is 0.5 mmol / L and the concentration of decitabine is 5 nmol / L, the expression level of HSA is the highest.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing human serum albumin using insect cells, characterized in that: Comprising the following steps: (1) Construct a donor vector containing the nucleotide sequence shown in SEQ ID NO. 1; (2) Transform the donor vector into DH10Bac competent cells to obtain recombinant bacmids; (3) Transfect the recombinant bacmids into insect cells to obtain a recombinant virus containing the nucleotide sequence of human serum albumin; (4) Infect insect cells with the recombinant virus and perform suspension serum-free culture; In step (1), the donor vector contains a signal peptide sequence added to the 5' end of the target gene; the nucleotide sequence of the signal peptide is as shown in SEQ ID NO. 2; in step (4), sodium butyrate and decitabine are added during the suspension serum-free culture; the insect cells are Sf9.

2. The method for producing human serum albumin using insect cells according to claim 1, wherein: In step (4), the final concentrations of sodium butyrate and decitabine added are 0.5 - 10 mmol / L and 5 - 50 nmol / L, respectively.

3. The method for producing human serum albumin using insect cells according to claim 1, characterized in that: In step (4), when performing the suspension serum-free culture, the medium used is Insect Pro serum-free medium.

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