C1 esterase inhibitor recombinant variant as well as preparation method and application thereof

Abstract

The invention discloses a C1 esterase inhibitor recombinant variant as well as a preparation method and application thereof. The amino acid sequence of the C1 esterase inhibitor recombinant variant is as shown in SEQ ID NO. 1. According to the present invention, the C1 esterase inhibitor recombinant variant is subjected to special gene modification, such that the vast majority of the functional active fragments are retained while the partial disordered region is deleted in the amino acid sequence optimization aspect so as to easily improve the expression quantity of the C1 esterase inhibitor recombinant variant protein; the gene sequence is subjected to cell preference codon optimization, and the C1 esterase inhibitor recombinant variant protein with high purity and higher enzyme activity is obtained. Meanwhile, the recombinant human serum albumin with a certain concentration is added in the preparation method, so that the success rate of single cell amplification can be obviously improved, the obtaining rate of a single cell strain is further improved, and the problems of low proliferation activity and difficulty in amplification of suspended single cells are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant variant of a C1 esterase inhibitor and a preparation method and application thereof. Background Art

[0002] C1 esterase inhibitor (C1-INH), also known as C1 inhibitor, is the only known plasma protease inhibitor that can regulate the complement system through both the classical pathway and the lectin pathway. It plays an important regulatory role in the complement system, kallikrein-kinin system, fibrinolytic system and coagulation system. It is mainly synthesized in the liver, and its deficiency is associated with hereditary angioneurotic edema (HANE). The average concentration in normal plasma is 0.2 mg / ml, and the C1-INH content in 1 ml of human plasma is equivalent to 1 IU of C1-INH. The main sources of C1 esterase inhibitors are plasma extraction and recombinant expression. Due to the limited source of plasma extraction and the susceptibility of blood sources to contamination by infectious diseases, the supply is relatively tight and the safety is not high. Therefore, the recombinant expression of human C1 esterase inhibitors provides the possibility of obtaining more C1-INH.

[0003] The expression of existing recombinant human C1 esterase inhibitors is generally achieved by transfecting plasmid vectors into mammalian cells, but they generally have the problems of low expression level and low activity of C1 esterase inhibitors.

[0004] Furthermore, many biological drugs, including monoclonal antibodies, are expressed and produced through the culture of mammalian cells. In order to ensure the stability of cells and the quality of products, single-cell cloning culture and proliferation are required in the early stages of product development. However, single or small numbers of cells are not easy to survive and reproduce, which poses great technical difficulties. Summary of the invention

[0005] In view of the defects in the prior art, the present invention provides a recombinant variant of a C1 esterase inhibitor and a preparation method and application thereof.

[0006] In order to solve the above technical defects, the present invention adopts the following technical solution: In a first aspect, a recombinant variant of a C1 esterase inhibitor is provided, wherein the amino acid sequence of the recombinant variant of the C1 esterase inhibitor is shown in SEQ ID NO.1.

[0007] In a second aspect, a gene encoding a recombinant variant of the C1 esterase inhibitor as described in the first aspect, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0008] In a third aspect, a method for preparing a recombinant variant of a C1 esterase inhibitor comprises the following steps: (1) connecting the nucleotide sequence described in the second aspect to the plasmid backbone and transferring it into competent cells to obtain a recombinant plasmid; (2) Introducing the recombinant plasmid of step (1) into host cells, and performing drug screening and monoclonal cell screening and amplification; (3) culturing the monoclonal cells obtained in step (2) in a fed-batch manner; (4) collecting the culture supernatant of step (3), performing ultrafiltration concentration, and purification to obtain the recombinant variant of the C1 esterase inhibitor; The step (2) includes the step of using a cell culture medium containing recombinant human serum albumin to perform suspension single cell expansion culture.

[0009] Furthermore, in step (2), the mass concentration of recombinant human serum albumin in the cell culture medium is 1-3 g / L; or in step (2), the mass concentration of recombinant human serum albumin in the cell culture medium is 1.5-2.5 g / L.

[0010] Furthermore, in the fed-batch culture of step (3), the temperature was lowered to 34° C. after 4 days of culture.

[0011] Furthermore, in step (4), purification includes affinity chromatography and anion chromatography, and the elution pH value in the anion chromatography is set to 8.0.

[0012] Furthermore, the preparation method uses a transposon system, and the transposon system is PiggyBac.

[0013] Furthermore, the host cells in step (2) include any one or more of CHO cells, HEK293 cells, and Sf9 cells.

[0014] In a fourth aspect, a recombinant expression vector is provided, wherein the recombinant expression vector contains the nucleotide sequence described in the second aspect.

[0015] The fifth aspect is a recombinant cell line comprising the nucleotide sequence described in the second aspect.

[0016] In a sixth aspect, a composition comprises any one or more of a recombinant variant of a C1 esterase inhibitor, the nucleotide sequence described in the second aspect, the recombinant expression vector described in the fourth aspect, and the recombinant cell line described in the fifth aspect. The recombinant variant of the C1 esterase inhibitor is the recombinant variant of the C1 esterase inhibitor described in the first aspect or the recombinant variant of the C1 esterase inhibitor prepared by the preparation method described in the third aspect.

[0017] A seventh aspect is the use of the composition described in the sixth aspect in the preparation of a medicament for preventing or treating hereditary angioedema.

[0018] Compared with the prior art, the present invention has the following technical effects: (1) The recombinant variant of C1 esterase inhibitor (LA-C1INH) of the present invention has undergone special genetic modification, and in terms of amino acid sequence optimization, some disordered regions have been deleted while retaining most of the functional active fragments, which is conducive to increasing the expression level of the recombinant variant protein of C1 esterase inhibitor. The gene sequence has been optimized for cell-preferred codons, which has improved the expression efficiency of the recombinant variant protein of C1 esterase inhibitor, and has better activity than other recombinant proteins and C1-INH proteins extracted from animal sources, thereby obtaining a recombinant variant protein of C1 esterase inhibitor with high purity and higher enzyme activity.

[0019] (2) Single or small number of cells are not easy to survive and reproduce. In addition, some difficult-to-transfect sequences have low cell activity and weak growth capacity, which makes it technically difficult to amplify these single cells. In view of the problem that the proliferation activity of the improved LA-C1INH sequence after transfection into suspension cells is significantly weaker than that of the wild-type sequence, and single cell amplification is difficult, the inventors have improved the components of the suspension single cell amplification medium through a large number of experiments in the face of the above technical difficulties. Recombinant human serum albumin (rHSA) was added to the single cell amplification medium, which can effectively support the growth of cells with low cell density and solve the problem of low proliferation activity and difficulty in amplification of LA-C1INH suspension single cells. Adding a certain concentration of recombinant human serum albumin can significantly improve the success rate of single cell amplification and further improve the acquisition rate of single cell strains. At the same time, it can also avoid the problem of contamination due to exogenous factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 A technical roadmap for the preparation of the LA-C1INH recombinant variants of the present invention; Figure 2 The structure of the LA-C1INH recombinant variant of the present invention and the schematic diagram of the PiggyBac vector; Figure 3 This is the electrophoresis diagram of the restriction endonuclease digestion of the vector containing the LA-C1INH sequence of Example 1 of the present invention (lane 181); Figure 4This is a WB image of Example 3 of the present invention; the order of loading samples in lanes 1 to 4 is: marker, empty load, WT, LA-C1INH; Figure 5 The effect of different cooling temperatures on cell growth metabolism and protein expression in the LA-C1INH suspension single cell fed-batch culture process; Figure A shows the viable cell density; Figure B shows the cell viability; Figure C shows the C1INH protein expression; Figure D shows the osmotic pressure; Figure 6 This is a graph showing the purity of LA-C1INH protein in Example 3 of the present invention; Figure 7 This is the ELISA protein expression graph of Example 4 of the present invention; Figure 8 This is a graph showing the ELISA enzyme activity detection results of Example 4 of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0023] Please refer to Figure 1 , 2 The codon-optimized base sequence was synthesized and connected to the transposon expression plasmid backbone. The expression plasmid and auxiliary plasmid were transfected into CHO cells by liposomes for drug screening and monoclonal cell screening and construction. The culture supernatant of LA-C1INH with high concentration was expressed by fed-batch culture. Finally, the His tag and anion chromatography were used for purification to obtain the recombinant variant protein of C1 esterase inhibitor with high purity and higher enzyme activity.

[0024] Example 1 Optimization of protein sequence and plasmid construction of recombinant variants of C1 esterase inhibitor 1. Optimization of C1 esterase inhibitor recombinant variant protein sequence According to the amino acid sequence data of C1 esterase inhibitor (NP_001027466.1) protein in the NCBI database, the disordered region in the amino acid sequence of human C1 esterase inhibitor was identified by bioinformatics software, and the non-functional disordered region sequence was removed in combination with its functional domain to obtain a recombinant variant of C1 esterase inhibitor, whose amino acid sequence is shown in SEQ ID NO. 1. The amino acid sequence before deleting the disordered region is shown in SEQ ID NO. 2, and this amino acid sequence plus a 6×His tag is the wild-type (WT) control sequence, and the nucleotide sequence is shown in SEQ ID NO. 3.

[0025] The amino acid sequence after deleting the disordered region was optimized for CHO cell preferred codons. The codon optimization was performed using an online codon optimization tool, and the GC content was adjusted from 53.69% to 55.73%. The nucleotide sequence before optimization is shown in SEQ ID NO.4, and the nucleotide sequence after optimization is shown in SEQ ID NO.5.

[0026] 2. Construction of LA-C1INH modified protein plasmid Double-stranded DNA fragments of C1 esterase inhibitor wild-type WT and LA-C1INH protein codon-optimized genes were prepared by gene synthesis, and the fragments were cloned into the mammalian gene expression PiggyBac vector pPB[Exp]-EGFP / Puro-CAG>ORF_Stuffer using homology arm technology. The expression plasmid and the PiggyBac auxiliary plasmid pRP[Exp]-mCherry-CAG>hyPBase were then transformed into Escherichia coli DH5α cells, plated, and single clones were picked. After amplification, the plasmid was extracted using an endotoxin-free plasmid extraction kit, and the sequence was sequenced to confirm the correctness. The LA-C1INH plasmid restriction enzyme digestion agarose gel electrophoresis was used to identify the Figure 3 As shown, M is a DNA marker, and 181 is the sequence of the plasmid after double digestion with ApaLI and EcoRI.

[0027] Example 2 Transfection, drug screening and expansion of suspension CHO cells The LA-C1INH plasmid constructed in Example 1 was expressed in CHO cells, and the specific steps were as follows: 1. Cell transfection (introduction into host cells) (1) One day before transfection, culture the pre-expanded suspension CHO cells at 0.8×10 cells per well. 6 Inoculate the cells into a 12-well low-adsorption plate with a cell culture medium volume of 1.5 mL in each well and place in an incubator for culture. The transfection effect is best after the inoculated cells have been cultured for 18-24 hours. At this time, replace the cells to be transfected with new culture medium and temporarily place them in an incubator; (2) Mix 75 μL of Opti-MEM medium (serum-free medium) and 4 μL of liposome transfection reagent 1 (derived from Lipofectamine 3000 transfection kit, including liposome transfection reagent 1 and liposome transfection reagent 2) in centrifuge tube No. 1 and place it in a centrifuge tube rack for later use; (3) Mix 3 μg of the plasmid to be transfected (1.5 μg each of the expression plasmid and the auxiliary plasmid), 6 μL of liposome transfection reagent 2, and 150 μL of opti-MEM medium in centrifuge tube No. 2 and place it in a centrifuge tube rack for later use; (4) Mix the reagents in centrifuge tube No. 1 and centrifuge tube No. 2, mix well by pipetting, and incubate at room temperature for 15 minutes; (5) Add the incubated transfection mixture to the cell culture medium of the cells to be transfected, shake crosswise to mix well, and place in the incubator.

[0028] 2. Puromycin screening of stable cell lines (drug screening, monoclonal cell screening and expansion) (1) 24 hours after transfection, add 5 μg / mL of puromycin to the cells in each well and stop the administration until all negative controls die. Half of the cells after drug screening are used for passaging and the other half are used for genome extraction. Then use the identification primers for PCR and sequencing to identify whether the target sequence has been integrated into the host cells. According to the sequencing results and cell growth status, select the appropriate cell colony for the next monoclonal cell screening.

[0029] (2) Resuspend the cells identified by drug screening sequencing in culture medium. After cell counting, dilute the cells using the limiting dilution method and take 260 cells and add them to 45 mL of single-cell basal culture medium. Use a spray gun to add the mixed cells to three 96-well plates at 150 μL / well. After placing them in the incubator, observe under a microscope every day to find the wells with single cells and mark the cell division and proliferation.

[0030] The single cell amplification efficiency is low when using conventional single cell basal culture medium such as VegaCHO. Since it is difficult for a single or small number of cells to survive and reproduce, and some difficult-to-transfect sequences have low cell activity and weak growth capacity, the proliferation activity of the LA-C1INH sequence in the present invention after transfection into suspension cells is significantly weaker than that of the wild-type sequence, and it is technically difficult to amplify these single cells.

[0031] This embodiment creatively uses recombinant human serum albumin to effectively support the growth of low-density cells. Using a medium containing recombinant human serum albumin to culture low-density cells can successfully recover and expand the cells, and also avoids a series of regulatory issues caused by the use of animal-derived ingredients.

[0032] In order to solve the problem of difficulty in amplifying LA-C1INH single cells, different concentrations of recombinant human serum albumin were selected and added to the single cell basal culture medium. Suspended single cell amplification experiments were performed according to the (phosphate buffer, PBS) addition group and the recombinant human serum albumin addition group (1g / L, 2g / L, 3g / L). After two weeks of amplification culture, the number of wells in the well plates with different concentrations of albumin added that finally successfully amplified was counted (successful amplification was defined as cells growing to 2 / 3 of the volume). Microscope detection was used to record the cell proliferation of each experimental group. Each experimental group had three parallel replicates, and the results were averaged. Finally, the single cell amplification efficiency was calculated. The results are shown in Table 1: Table 1 Effects of different concentrations of recombinant human serum albumin on CHO single cell expansion From the above data, it can be seen that the number of successful wells of cell expansion in the three experimental groups with added recombinant human serum albumin was significantly higher than that in the PBS control group. Among them, the experimental group with added 2g / L recombinant human serum albumin had the highest success rate of LA-C1INH single cell expansion. This shows that adding 1~3g / L recombinant human serum albumin can more effectively promote the expansion of suspended single cells, and it is better to add 1.5~2.5g / L. The optimal recombinant human serum albumin concentration is 2g / L.

[0033] (3) Change the medium in the wells containing single cells every day. When the cells have grown to 2 / 3 of the area, subculture the cells to a larger well plate, and so on. When the single cells have been amplified to a sufficient number, take a portion of the genome for PCR and sequencing to identify whether the target sequence has been integrated into the host cell.

[0034] PCR verification primers: Check-F: SEQ ID NO.6; Check-R: SEQ ID NO.7.

[0035] Example 3 Identification and Fed-batch Culture of Recombinant Variants of C1 Esterase Inhibitor 1. Collect the supernatant of empty, WT, and LA-C1INH cells, add SDS loading buffer, and use Western blot to identify the size and specificity of C1-INH expression. The results are as follows Figure 4 As shown, both WT and LA-C1INH recombinant variant proteins can specifically bind to the C1 esterase inhibitor antibody, and the size of the LA-C1INH recombinant variant protein is half that of the WT.

[0036] 2. The single cell LA-C1INH high-expressing cell line obtained by limiting dilution amplification was cultured in a 1L shake flask for 16 days in a fed-batch format, and the culture supernatant was collected. 6Cells were seeded at a density of 10 cells / mL in vegaCHO medium (OPM, Cat. P121662) and cultured in a shaker at 37°C (humidity 80%, speed 130 rpm, 5% CO2). Starting from days 2 / 4 / 6 / 8 / 10 / 12 / 14, the cultures were supplemented with different feeds every day, including 3% / 4% / 5% / 5% / 4% / 3% by volume of VegaCHO Feed (OPM, Cat. P134305) and 0.3% / 0.4% / 0.5% / 0.5% / 0.4% / 0.3% by volume of CDFS36 until harvesting on day 16. On the fourth day of culture, the temperature was lowered to prolong the maintenance of the density peak. Glucose was added to the culture to maintain a culture concentration of 3-6 g / L. The culture supernatant was collected, centrifuged (2000g, 10 minutes), and stored at -20°C for subsequent analysis.

[0037] 3. In order to maximize the expression of LA-C1INH, the cooling culture temperature that affects the yield of CHO cell Fed-batch process was screened and optimized. 6 The cells were inoculated at a density of 10 cells / ml in a shake flask. After culturing for 4 days, the culture temperature was lowered from 37°C to 31°C, 32°C, 33°C, 34°C, and 35°C, respectively, and the culture was continued until the 16th day. During this period, samples were collected and tested every day to record the viable cell density, cell viability, osmotic pressure, and protein expression. The results are as follows Figure 5 As shown, after 4 days of culture, the temperature was lowered to 34°C. After 16 days of culture, the expression level of LA-C1INH reached the highest level, and the peak cell density was maintained for a relatively long time, thus achieving the best expression effect.

[0038] Example 4 Purification of recombinant variants of C1 esterase inhibitor and determination of supernatant expression and activity 1. After the collected cell supernatant was centrifuged at 1000 rpm and 4°C, the supernatant was filtered through a 0.22 μm filter membrane to remove impurities. The clarified solution was passed through a 10KD membrane package and concentrated 5-10 times. Finally, 20m Tris buffer was used for filtration. The clarified and filtered protein was affinity chromatographed with Ni-excel filler, the loading buffer contained 20 mM Tris, and the LA-C1INH protein was eluted with a 20-100 mM imidazole concentration gradient to obtain a purified LA-C1INH sample. In addition, in order to further improve the purity of LA-C1INH, anion chromatography was performed using a Capto Q column. We explored the optimal elution conditions and finally determined that pH 8.0 (20m Tris, 100mM) was the best elution pH for LA-C1INH. We tested the protein recovery and purity of different pH gradient eluents (see Table 2). We found that the LA-C1INH protein with a purity of nearly 97% could be obtained using pH 8.0 eluent (see Table 2). Figure 6 ), and can ensure a relatively high recovery rate.

[0039] Table 2 Statistical details of protein recovery and purity in chromatography process 2. Expression level of LA-C1INH protein The protein concentration of LA-C1INH in the cell supernatant was determined using an ELISA kit (abcam, ab224883) according to the instructions, and the expression level of LA-C1INH in the recombinant host cells was calculated.

[0040] The results are as follows Figure 7 As shown, compared with WT, the expression level of LA-C1INH protein was significantly increased, and the expression amount could reach 600 mg / L.

[0041] 3. Activity determination of LA-C1INH protein The activity of LA-C1INH in the cell supernatant was determined using the STAGO fully automatic coagulation instrument, referring to the method in the article "Establishment and Validation of Dynamic Colorimetric Detection Method for Human C1 Esterase Inhibitor Activity" published in the Chinese Journal of Biological Products. The results are shown in Figure 8 .

[0042] The results showed that the activity of the purified sample of the LA-C1INH recombinant variant was significantly improved compared with that of the WT, and had good activity.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant variant of a C1 esterase inhibitor, characterized in that The amino acid sequence of the recombinant variant of the C1 esterase inhibitor is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant variant of a C1 esterase inhibitor as claimed in claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.4 or SEQ ID NO.

5.

3. A method for preparing a recombinant variant of a C1 esterase inhibitor, characterized in that: The steps include: (1) connecting the nucleotide sequence described in claim 2 to a plasmid backbone and transferring the resultant nucleotide sequence into competent cells to obtain a recombinant plasmid; (2) Introducing the recombinant plasmid of step (1) into host cells, and performing drug screening and monoclonal cell screening and amplification; (3) culturing the monoclonal cells obtained in step (2) in a fed-batch manner; (4) collecting the culture supernatant of step (3), performing ultrafiltration concentration, and purification to obtain the recombinant variant of the C1 esterase inhibitor; The step (2) includes the step of using a cell culture medium containing recombinant human serum albumin to perform suspension single cell expansion culture.

4. The method for preparing a recombinant variant of a C1 esterase inhibitor according to claim 3, characterized in that: In step (2), the mass concentration of recombinant human serum albumin in the cell culture medium is 1-3 g / L; or in step (2), the mass concentration of recombinant human serum albumin in the cell culture medium is 1.5-2.5 g / L.

5. The method for preparing a recombinant variant of a C1 esterase inhibitor according to claim 3, characterized in that: In the fed-batch culture of step (3), the temperature was lowered to 34°C after 4 days of culture.

6. The method for preparing a recombinant variant of a C1 esterase inhibitor according to claim 3, characterized in that: In step (4), purification includes affinity chromatography and anion chromatography, and the elution pH value in the anion chromatography is set to 8.

0.

7. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleotide sequence according to claim 2.

8. A recombinant cell line, characterized in that Containing the nucleotide sequence of claim 2.

9. A composition, characterized in that The composition comprises any one or more of a recombinant variant of a C1 esterase inhibitor, a nucleotide sequence according to claim 2, a recombinant expression vector according to claim 7, and a recombinant cell line according to claim 8; the recombinant variant of the C1 esterase inhibitor is a recombinant variant of the C1 esterase inhibitor according to claim 1 or a recombinant variant of the C1 esterase inhibitor prepared by the preparation method according to claim 3.

10. Use of the composition according to claim 9 in the preparation of a medicament for preventing or treating hereditary angioedema.

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