Expression vector, expression system and preparation method of recombinant larosidase protein

Abstract

The invention provides an expression vector, an expression system and a preparation method of a recombinant larosidase protein, and belongs to the technical field of recombinant protein preparation, the recombinant expression vector of the larosidase protein provided by the invention comprises an initial vector and a codon-optimized human-derived larosidase coding gene; the nucleotide sequence of the human-derived larosidase coding gene is as shown in SEQ ID NO. 1; according to the recombinant expression system, Chinese hamster ovary cells are taken as host cells, and the recombinant expression vector is transfected. According to the invention, a gene sequence more suitable for expression of Chinese hamster ovary cells is obtained through codon optimization, and then transposon-mediated stable integration is carried out; high-density suspension culture and downstream purification processes are utilized to realize efficient and stable expression of the recombinant larosidase in CHO cells, and the recombinant larosidase with high yield, high activity and high purity can be obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of recombinant protein preparation, and particularly relates to an expression vector and an expression system of recombinant laronidase protein and a preparation method thereof. BACKGROUND

[0002] Mucopolysaccharidosis type I is a rare autosomal recessive disease caused by the deletion of enzyme activity due to mutations in the alpha-L-iduronidase (i.e. laronidase) gene. Enzyme replacement therapy is the current main treatment, and the core is to infuse the recombinant laronidase protein with biological activity into the patient's body.

[0003] Chinese hamster ovary (CHO) cells are one of the most recognized safe and most suitable host systems for industrial large-scale production of recombinant therapeutic proteins. Its advantages include: it can realize high-density suspension culture, has good scalability, does not carry human pathogenic viruses, and has similar post-translational modification capabilities (such as glycosylation) to human cells.

[0004] As a lysosomal enzyme, the complex post-translational modification (such as correct glycosylation pattern) of laronidase is the key to realize its targeted delivery to lysosomes and function, and the expression level of laronidase gene with biological activity in CHO cells is difficult to guarantee by a simple expression system; therefore, it is urgent to develop a recombinant expression system and preparation process optimized for CHO cells, which can efficiently express and ensure the correct folding and modification of laronidase, and reduce production costs. SUMMARY

[0005] Therefore, the purpose of the present application is to provide an expression vector and an expression system of recombinant laronidase protein and a preparation method thereof, which realizes stable and high-yield expression of recombinant laronidase by using a codon-optimized gene on a specific expression vector.

[0006] The present application provides a recombinant expression vector of laronidase protein, which comprises an initial vector and a codon-optimized human laronidase coding gene; the nucleotide sequence of the human laronidase coding gene is shown in SEQ ID NO. 1.

[0007] Preferably, the initial vector carries a strong promoter CMV and a transposon element derived from the piggyBac transposon system.

[0008] Preferably, the human laronidase coding gene is seamlessly cloned between the CMV promoter and the WPRE element.

[0009] Preferably, the initial vector is a Freedom® pCHO1.0 vector.

[0010] The application provides a recombinant expression system of laronidase protein, which is a host cell of Chinese hamster ovary cells and is transfected with the recombinant expression vector.

[0011] The application provides a preparation method of recombinant laronidase, which comprises the following steps: S1) transfecting the recombinant expression vector into Chinese hamster ovary cells to obtain a recombinant CHO cell pool through screening; S2) performing limited dilution on the recombinant CHO cell pool to obtain a single clone expressing recombinant laronidase protein; S3) performing large-scale culture on the obtained single clone; S4) separating and purifying the recombinant laronidase protein from the supernatant obtained through culture.

[0012] Preferably, the large-scale culture in step S3) is high-density suspension culture.

[0013] Preferably, the purification is selected from one or more of affinity chromatography, ion exchange chromatography and molecular exclusion chromatography.

[0014] The application provides application of the recombinant expression vector and the recombinant expression system in preparation of a medicine for treating mucopolysaccharidosis type I.

[0015] Compared with the prior art, the application has the following beneficial effects: the application provides a recombinant expression vector of laronidase protein, which comprises an initial vector and a codon-optimized human laronidase coding gene, and the application obtains a gene sequence more suitable for expression of Chinese hamster ovary cells through codon optimization, and then obtains a recombinant expression system with good stability and high yield through transposon-mediated stable integration.

[0016] Further, the application realizes high-efficiency and stable expression of recombinant laronidase in CHO cells through high-density suspension culture and downstream purification process, and high-yield, high-activity and high-purity recombinant laronidase can be obtained. The preparation method of the recombinant laronidase provided by the application is safe, efficient and easy to scale up, and can realize large-scale production of enzymes for treating mucopolysaccharidosis type I. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a structural schematic diagram of the recombinant expression vector.

[0018] Figure 2 It is a result diagram of verification of expression of recombinant laronidase connected with different promoters (CMV and HSP90) in CHO cells through Western Blot.

[0019] Figure 3To screen the high-efficiency secretory expression of recombinant laronidase protein monoclonal cell strain identification, wherein M is marker, and the rest is different monoclonal cell strain.

[0020] Figure 4 To the PAGE result figure of the purified recombinant laronidase protein in the embodiment of the application, wherein M is marker, 100, 200, 300, 500, 1M are different concentrations of imidazole eluent. DETAILED DESCRIPTION

[0021] The application provides a recombinant expression vector of laronidase protein, which comprises an initial vector and a codon-optimized human laronidase encoding gene; the nucleotide sequence of the human laronidase encoding gene is shown in SEQ ID NO. 1, and specifically as follows: In the present application, the initial vector carries a strong promoter CMV and a transposon element derived from piggyBac transposon system; the strong promoter CMV is preferably cytomegalovirus immediate early promoter (CMV-IE) for driving high level transcription of laronidase gene; the inverted terminal repeat (ITR) in the piggyBac transposon system is for stably integrating laronidase gene into host genome; preferably, the initial vector also carries a selectable marker gene, a Blasticidin resistance gene, for screening stably transformed cell lines. In the present application, the initial vector is preferably Freedom® pCHO1.0 vector; the human laronidase coding gene is preferably seamlessly cloned between CMV promoter and WPRE element.

[0022] The present application also provides a recombinant expression system of laronidase protein, which is a host cell of Chinese hamster ovary cell transfected with the recombinant expression vector. The present application does not have special limitations on the preparation method of the recombinant expression system, and the commonly known recombinant cell preparation method in the art can be used.

[0023] The present application provides a preparation method of recombinant laronidase, comprising the following steps: S1) transfecting the recombinant expression vector into Chinese hamster ovary cells to obtain a recombinant CHO cell pool; S2) performing limited dilution on the recombinant CHO cell pool to obtain a single clone expressing recombinant laronidase protein; S3) expanding culture of the obtained single clone; and S4) separating and purifying the recombinant laronidase protein from the supernatant obtained by culture.

[0024] In the present application, the recombinant expression vector is transfected into Chinese hamster ovary cells to obtain a recombinant CHO cell pool. The recombinant expression vector is preferably obtained by recombining the human laronidase coding gene into the initial vector; further preferably, the seamless cloning technology is used to insert the human laronidase coding gene between the CMV promoter and the downstream WPRE element of the Freedom® pCHO1.0 vector; and the human laronidase coding gene is preferably optimized for Chinese hamster codon bias. The present application transfects the prepared recombinant expression vector into Chinese hamster ovary cells. The present application does not have special limitations on the transfection method, and the conventional cell transfection method in the art can be used.

[0025] In the present application, the recombinant CHO cell pool is obtained, and the recombinant CHO cell pool is subjected to limited dilution to obtain a single clone expressing recombinant laronidase protein; the present application preferably performs pressure screening after transfection, and the pressure screening is preferably achieved by adding BSD in the culture medium, and then western blot is used to identify the expression of recombinant laronidase protein.

[0026] The present application, after obtaining the monoclonal expressing recombinant laronidase protein, screens the monoclonal with high expression amount of recombinant laronidase protein according to the expression amount of recombinant laronidase protein for next step of expansion culture. In the present application, the concentration of inoculated cells in the expansion culture is preferably 3-8×10 5 cells / mL, and more preferably 5×10 5 cells / mL; the expansion culture is preferably cultured in a 37℃, 5% CO2 cell incubator, and the rotation speed is preferably 100-150 rpm, and more preferably 110-130 rpm.

[0027] After the expansion culture, the recombinant laronidase protein is separated and purified from the supernatant obtained by culture; the purification is selected from one or more of affinity chromatography, ion exchange chromatography and molecular exclusion chromatography.

[0028] The present application also provides the use of the recombinant expression vector and the recombinant expression system in the preparation of a medicament for treating mucopolysaccharidosis type I.

[0029] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0030] Example 1

[0031] Construction of recombinant laronidase protein expression vector

[0032] According to the codon usage frequency of CHO cells, the coding sequence of human laronidase is codon-optimized and gene-synthesized (the sequence is shown as SEQ ID NO. 1). The sequence is inserted into the Freedom®pCHO1.0 vector (Increase recombinant antibody yields through optimizing vector design and production process in CHO cells) at 2544 bp, adjacent to the upstream CMV promoter and downstream WPRE element, without additional base residues, to achieve a truly "seamless" insertion, and the final recombinant expression vector structure is shown in Figure 1 . The expression vector is artificially synthesized by General Biosystems Co., Ltd. (Chuzhou, China).

[0033] The control expression vector uses HSP90 promoter, and the rest is the same.

[0034] Example 2

[0035] Expression and purification of recombinant laronidase protein

[0036] Establishment of cell strain expressing recombinant laronidase protein

[0037] (1) Cell culture: CHO-S cells (#A11557-01; Carlsbad, CA, USA) were cultured in DMEM / F12 complete medium containing 10% inactivated fetal bovine serum at 37°C, 5% CO2, to the logarithmic growth phase, and the cells were collected by trypsinization. The required cell suspension volume was calculated according to the density of 2 x 10 5 / mL, and was added dropwise to a 24-well plate. Then 1 mL of prepared DMEM / F12 complete medium was added for further culture. When the cell confluence reached about 70%-80%, the cells were used for transfection.

[0038] (2) Cell transfection: The recombinant expression vector and control expression vector constructed in Example 1 were respectively transfected according to the instructions of lipofectamine 2000 transfection reagent. 4 μL of lipo2000 and 1.6 μg of recombinant expression vector (or control expression vector) were added to 100 μL of DMEM / F12 medium and mixed. After 5 min, the mixed transfection reagent diluent and recombinant expression vector diluent were mixed and incubated at room temperature for 20 min. The culture medium in the cell culture well was removed, 1 mL of DMEM / F12 complete medium was added to each well, and the complex containing the transfection reagent and the recombinant expression vector was added to the culture well and incubated in the incubator. After 4-6 h, fresh complete medium was replaced for further culture.

[0039] (3) Stable cell strain screening: 48 h after transfection, pressure screening was started: the six-well plate cells were taken out from the 37°C incubator, the supernatant medium was discarded, and 2 mL of DMEM / F12 (containing 10% serum + 15 μg / mL BSD) was added. The pressure screening was continued until the negative control cells were basically dead. The BSD concentration was reduced to 10 μg / mL, and the culture was continued until the cell pool grew at the same speed as the blank CHO cells.

[0040] (4) Recombinant laronidase protein expression identification

[0041] The stable cell strain screened after transfection was collected, and western blot was used to identify the laronidase protein: after protein separation by polyacrylamide gel electrophoresis, the protein was transferred to a PVDF membrane by wet transfer method, and 5% skimmed milk powder was blocked at room temperature for 1 h 30 min. Diluted Anti-HIS Tag Antibody was added, and the shaking incubator was incubated at 4°C overnight. The membrane was washed with TBST for 10 min x 3 times, and Mouse Anti-Human IgG diluent was added and incubated at room temperature for 1 h. The membrane was washed with TBST for 10 min x 3 times, and luminescence solution A and luminescence solution B were mixed at a ratio of 1:1 and used immediately. The prepared luminescence solution was added to the washed membrane in the gel imaging system, and then exposed and developed. Figure 2 The results of detecting the expression of recombinant laronidase protein in culture supernatant are shown. The recombinant expression vector with CMV promoter was used to transfect cells, and the expression amount of recombinant laronidase protein was significantly higher than that of control SHP90.

[0042] (5) Establishment of a monoclonal cell strain of recombinant laronidase protein

[0043] The monoclonal cell strain expressing recombinant laronidase protein was obtained by limiting dilution method. The monoclonal cell strain was cultured, trypsinized, and the cells were suspended in serum-free medium and counted. The cells were diluted to 5×10 5 cells / mL and inoculated in 4 mL culture medium in a 6-well plate. The cell density and viability were counted every 24 h. After the culture ended, the supernatant was treated and the expression comparison results (results are shown in Figure 3 ).

[0044] Expression, purification and identification of recombinant laronidase protein

[0045] The monoclonal cell strain D27 expressing recombinant laronidase protein was cultured as described above. The cells were taken out of the 37°C incubator, the supernatant medium was discarded, and the cells were washed once with pre-warmed 8 mL PBS and the PBS was discarded. 1-2 mL trypsin was added to each 10 cm cell culture dish, and the cells were observed under a microscope to be shrunk and rounded, and single cells were added with 4 mL DMEM / F12 (containing 10% serum, 10 μg / mL BSD) to terminate digestion and the cells were blown away with a pipette. The digested cells were transferred to a 15 mL centrifuge tube, centrifuged at room temperature at 1000 rpm for 5 min. The cells were suspended in serum-free medium and counted. The cells were diluted to 5×10 5The 150 mL cell suspension was inoculated in a 500 mL flask. The cell culture flask was placed on a orbital shaker in a 37℃, 5% CO2 incubator at 120 rpm overnight. The surface of the biosafety cabinet was disinfected with 75% alcohol and UV irradiation for 30 min. The cell density and viability were counted every 24 h. After the end of the culture, the supernatant was taken for subsequent purification.

[0046] 2 mL Ni-NTA Sepharose slurry (50% suspension) was packed in a column, and 150 mL of the supernatant (obtained by cell culture) was loaded after equilibration with imidazole binding buffer (20 mM NaH2PO4, 0.5 M NaCl, 25 mM imidazole) or PBS (pH 7.4) for 5 column volumes. After equilibration again, elution was performed with 2 mL eluent of different concentrations of imidazole (100 mM, 200 mM, 300 mM, 500 mM, 1 M), respectively. After elution, equilibration was performed again, and 5 column volumes of deionized water were washed, and then 20% ethanol was added for storage at 4℃.

[0047] The prepared recombinant laronidase protein was identified by SDS-PAGE: after protein separation by polyacrylamide gel electrophoresis, Coomassie brilliant blue staining was used for slow shaking on a shaker for 30 min, and then the decolorizing solution was shaken on a shaker every 1-2 h, and the decolorizing solution was replaced until the background became transparent and the protein band was clearly visible. The recombinant laronidase was obtained by purification, and the size of the purified laronidase protein was clearly shown (as shown in the results of Figure 4

[0048] Example 3 Protein concentration determination

[0049] The BCA kit (P0010S, Biyun Tian Biotechnology Co., Ltd.) was used to determine that the protein amount in the supernatant obtained by cell culture after 7 days of cell culture in 0.15 L was 10 mg, so the protein amount in 1 L of culture supernatant was 67 mg, and the protein yield was 67 mg / L.

[0050] From the above examples, it can be seen that the expression vector, expression system and preparation method of the recombinant laronidase protein realize stable and high-yield expression of the recombinant laronidase by using the codon-optimized gene on a specific expression vector.

[0051] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.​

Claims

1. A recombinant expression vector for laronidase protein, characterized in that, The invention includes an initial vector and a codon-optimized human laronidase encoding gene; the nucleotide sequence of the human laronidase encoding gene is shown in SEQ ID NO.

1.

2. The recombinant expression vector according to claim 1, characterized in that, The initial carrier carries a strong starter CMV and a transposable element derived from the piggyBac transposable subsystem.

3. The recombinant expression vector according to claim 1 or 2, characterized in that, The human laronidase-encoding gene was seamlessly cloned between the CMV promoter and the WPRE element.

4. The recombinant expression vector according to claim 3, characterized in that, The initial vector was the Freedom® pCHO1.0 vector.

5. A recombinant expression system for laronidase protein, characterized in that, The recombinant expression system uses Chinese hamster ovary cells as host cells and is transfected with the recombinant expression vector described in any one of claims 1 to 4.

6. A method for preparing recombinant laronidase, characterized in that, Includes the following steps: S1) Transfect Chinese hamster ovary cells with the recombinant expression vector according to any one of claims 1 to 4, and screen to obtain a recombinant CHO cell pool; S2) The recombinant CHO cell pool was subjected to limiting dilution to obtain single clones expressing recombinant laronidase protein; S3) Expand the single clones obtained above into larger-scale cultures; S4) The recombinant laronidase protein was separated and purified from the supernatant obtained from the culture.

7. The preparation method according to claim 6, characterized in that, Step S3) describes a high-density suspension culture.

8. The preparation method according to claim 6, characterized in that, The purification process is selected from one or more of affinity chromatography, ion exchange chromatography, and size exclusion chromatography.

9. The use of the recombinant expression vector according to any one of claims 1 to 4 and the recombinant expression system according to claim 5 in the preparation of a medicament for treating mucopolysaccharidosis type I.

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