A recombinant variant of C1 esterase inhibitor, its preparation method and applications

Abstract

The present invention discloses a recombinant variant of C1 esterase inhibitor, its preparation method and application. A recombinant variant of C1 esterase inhibitor has an amino acid sequence as shown in SEQ ID NO.1. The recombinant variant of C1 esterase inhibitor of the present invention undergoes special genetic modification. While deleting part of the disordered region in the amino acid sequence optimization, most of the functional active fragments are retained, which is beneficial to improving the expression level of the recombinant variant protein of C1 esterase inhibitor; the gene sequence is optimized with cell-preferred codons to obtain a recombinant variant protein of C1 esterase inhibitor with high purity and higher enzyme activity. At the same time, adding a certain concentration of recombinant human serum albumin in the preparation method of the present invention can significantly improve the success rate of single-cell amplification, further improve the acquisition rate of single-cell strains, and solve the problems of low proliferation activity and difficult amplification of suspended single cells.

Description

Technical Field

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

[0002] C1 esterase inhibitor (C1-INH), also known as C1 inhibitor, is the only plasma protease inhibitor known so far 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 related to hereditary angioedema (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 inhibitor are plasma extraction and recombinant expression. Due to the limited source of plasma extraction and the easy contamination of blood sources by infectious diseases, the supply is relatively tight and the safety is not high. Therefore, the recombinant expression of human C1 esterase inhibitor provides the possibility of obtaining more C1-INH.

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

[0004] Furthermore, many biological drugs, including monoclonal antibodies, are expressed and produced by culturing mammalian cells. In order to ensure the stability of cells and the quality of products, the culture and proliferation of single-cell clones need to be carried out at the initial stage of product research and development. However, single or a small number of cells are not easy to survive and reproduce, and there are great technical difficulties. Summary of the Invention

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

[0006] To solve the above technical defects, the present invention adopts the following technical solutions:

[0007] In the first aspect, a recombinant variant of C1 esterase inhibitor, wherein the amino acid sequence of the recombinant variant of C1 esterase inhibitor is as shown in SEQ ID NO.1.

[0008] In the second aspect, a gene encoding the recombinant variant of C1 esterase inhibitor as described in the first aspect, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0009] In a third aspect, a method for preparing a recombinant variant of C1 esterase inhibitor includes the following steps:

[0010] (1) Connect the nucleotide sequence described in the second aspect above to a plasmid backbone, transfer it into competent cells to obtain a recombinant plasmid;

[0011] (2) Introduce the recombinant plasmid in step (1) into a host cell, and perform drug screening and monoclonal cell screening and amplification;

[0012] (3) Perform fed-batch culture on the monoclonal cells in step (2);

[0013] (4) Collect the culture supernatant in step (3), perform ultrafiltration concentration and purification to obtain the recombinant variant of C1 esterase inhibitor;

[0014] In step (2), it includes the step of performing suspension single-cell amplification culture using a cell culture medium containing recombinant human serum albumin.

[0015] Further, 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.

[0016] Further, in the fed-batch culture in step (3), the temperature is reduced to 34 °C after 4 days of culture.

[0017] Further, in step (4), the purification includes affinity chromatography and anion chromatography, and the elution pH value in the anion chromatography is set to 8.0.

[0018] Further, the transposon system used in the preparation method is the PiggyBac transposon system.

[0019] Further, the host cell in step (2) includes any one or more of CHO cells, HEK293 cells, and Sf9 cells.

[0020] In a fourth aspect, a recombinant expression vector contains the nucleotide sequence described in the second aspect above.

[0021] In a fifth aspect, a recombinant cell line contains the nucleotide sequence described in the second aspect above.

[0022] Sixth aspect, a composition, which comprises any one or more of a C1 esterase inhibitor recombinant variant, the nucleotide sequence described in the second aspect above, the recombinant expression vector described in the fourth aspect above, and the recombinant cell line described in the fifth aspect above. The C1 esterase inhibitor recombinant variant is the C1 esterase inhibitor recombinant variant described in the first aspect or the C1 esterase inhibitor recombinant variant prepared by the preparation method described in the third aspect.

[0023] Seventh aspect, application of the composition described in the sixth aspect above in the preparation of a drug for preventing or treating hereditary angioedema.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] (1) The C1 esterase inhibitor recombinant variant (LA-C1INH) of the present invention has undergone special genetic modification. While deleting part of the disordered region in the amino acid sequence optimization, most of the functional active fragments are retained, which is beneficial to improving the expression level of the C1 esterase inhibitor recombinant variant protein. The gene sequence has been optimized with cell-preferred codons, improving the expression efficiency of the C1 esterase inhibitor recombinant variant protein, and having better activity compared with other recombinant proteins and C1-INH proteins extracted from animals, obtaining a C1 esterase inhibitor recombinant variant protein with high purity and higher enzyme activity.

[0026] (2) Single or a small number of cells are not easy to survive and reproduce. Coupled with the low cell activity and weak proliferation ability of some cells with difficult-to-transfect sequences, there are great technical difficulties in amplifying these single cells. In view of the problem that the proliferation activity of the improved LA-C1INH sequence transfected into suspension cells is significantly weaker than that of the wild-type sequence and the difficulty in single-cell amplification, when the inventors faced the above technical problems, they improved the composition of the suspension single-cell amplification medium through a large number of experiments. Recombinant human serum albumin (rHSA) was added to the single-cell amplification medium, which can effectively support the growth of cells at low cell density, solve the problems of low proliferation activity and difficult amplification of LA-C1INH suspension single cells, and adding a certain concentration of recombinant human serum albumin can significantly improve the success rate of single-cell amplification and further increase the acquisition rate of single-cell strains. At the same time, it can also avoid the problem of exogenous factor contamination. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 This is the technical roadmap for the preparation of the LA-C1INH recombinant variant of the present invention;

[0029] Figure 2 This is a schematic diagram of the structure of the LA-C1INH recombinant variant of the present invention and the PiggyBac vector;

[0030] Figure 3 This is the restriction enzyme digestion electrophoresis map of the vector containing the LA-C1INH sequence in Example 1 of the present invention (lane 181);

[0031] Figure 4 This is the WB map of Example 3 of the present invention; The loading order of lanes 1-4 is: marker, empty vector, WT, LA-C1INH;

[0032] Figure 5 In the fed-batch culture process of LA-C1INH suspension single cells, the effects of different cooling temperatures on cell growth metabolism and protein expression level; In the figure, Figure A is the viable cell density; Figure B is the cell viability; Figure C is the C1INH protein expression level; Figure D is the osmotic pressure;

[0033] Figure 6 This is the protein purity detection map of LA-C1INH in Example 3 of the present invention;

[0034] Figure 7 This is the ELISA protein expression level map of Example 4 of the present invention;

[0035] Figure 8 This is the ELISA enzyme activity detection result map of Example 4 of the present invention. Detailed implementation manners

[0036] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] Please refer to Figure 1 、 2, the codon-optimized base sequence was gene-synthesized and ligated onto the transposon expression plasmid backbone. The expression plasmid and the helper plasmid were transfected into CHO cells by liposome for drug screening and monoclonal cell screening construction. High-concentration LA-C1INH culture supernatant was expressed through fed-batch culture. Finally, purification was carried out using His-tag and anion chromatography to harvest the recombinant variant protein of C1 esterase inhibitor with high purity and higher enzyme activity.

[0038] Example 1 Sequence optimization and plasmid construction of recombinant variant protein of C1 esterase inhibitor

[0039] 1. Sequence optimization of recombinant variant protein of C1 esterase inhibitor

[0040] According to the protein amino acid sequence data of C1 esterase inhibitor (NP_001027466.1) in the NCBI database, the disordered regions in the amino acid sequence of human C1 esterase inhibitor were identified through bioinformatics software. The non-functional disordered region sequences were removed in combination with its functional domains to obtain the recombinant variant of C1 esterase inhibitor, and its 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 with a 6×His tag is the wild-type (WT) control sequence, and the nucleotide sequence is shown in SEQ ID NO.3.

[0041] The amino acid sequence after deleting the disordered region was optimized for CHO cell-preferred codons. Codon optimization was carried out through 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 SEQID NO.4, and the optimized nucleotide sequence is shown in SEQ ID NO.5.

[0042] 2. Construction of LA-C1INH recombinant plasmid

[0043] Double-stranded DNA fragments of the wild-type WT of C1 esterase inhibitor and the gene with codon-optimized LA-C1INH protein were prepared by gene synthesis. The fragments were cloned onto the mammalian gene expression PiggyBac vector pPB[Exp]-EGFP / Puro-CAG>ORF_Stuffer using homologous arm technology. Subsequently, the expression plasmid and the PiggyBac helper plasmid pRP[Exp]-mCherry-CAG>hyPBase were transformed into Escherichia coli DH5α cells, plated, single colonies were picked, amplified, and plasmids were extracted using an endotoxin-free plasmid extraction kit and sequenced to confirm the correctness of the sequence. The restriction enzyme digestion agarose gel electrophoresis identification of the LA-C1INH plasmid is as Figure 3 shown, M is the DNA Marker, and 181 is the sequence after double digestion of the plasmid with ApaLI and EcoRI.

[0044] Example 2: Transfection, Drug Screening and Amplification of Suspended CHO Cells

[0045] The LA-C1INH plasmid constructed in Example 1 was expressed in CHO cells, and the specific steps were as follows:

[0046] 1. Cell Transfection (Introduction into Host Cells)

[0047] (1) One day before transfection, the pre-amplified suspended CHO cells were inoculated into a 12-well low-attachment plate at a density of 0.8×10 6 cells per well, with a volume of 1.5 mL of cell culture medium in each well, and placed in an incubator for culture. The transfection effect was the best when the inoculated cells were cultured for 18 - 24 hours. At this time, the cells to be transfected were replaced with fresh medium and temporarily placed in the incubator;

[0048] (2) 75 μL of opti-MEM medium (serum-free medium) and 4 μL of liposome transfection reagent 1 (derived from the Lipofectamine 3000 transfection kit, including liposome transfection reagent 1 and liposome transfection reagent 2) were thoroughly mixed in a centrifuge tube No. 1 and placed on a centrifuge tube rack for standby;

[0049] (3) 3 μg of the plasmid to be transfected (1.5 μg each of the expression plasmid and the helper plasmid), 6 μL of liposome transfection reagent 2 and 150 μL of opti-MEM medium were thoroughly mixed in a centrifuge tube No. 2 and placed on a centrifuge tube rack for standby;

[0050] (4) The reagents in centrifuge tube No. 1 and centrifuge tube No. 2 were mixed, and pipetted to mix evenly, and incubated at room temperature for 15 minutes;

[0051] (5) The incubated transfection mixture was added to the cell culture medium of the cells to be transfected, shaken crosswise, and placed in an incubator.

[0052] 2. Screening of Stable Transfected Cell Lines with Puromycin (Drug Screening, Monoclonal Cell Screening and Amplification)

[0053] (1) 24 hours after transfection, 5 μg / mL of puromycin was added to the cells in each well for treatment until all the negative controls died. Half of the cells after drug screening were used for passage, and half were used for genomic DNA extraction. Then PCR was performed with identification primers, and sequencing was used to identify whether the target sequence was integrated into the host cells. According to the sequencing results and the cell growth status, appropriate cell colonies were selected for the next step of monoclonal cell screening.

[0054] (2) Resuspend the cells identified by drug screening sequencing with the culture medium. After cell counting, dilute the cells using the limited dilution method. Take 260 cells and add them to 45 mL of single-cell basal medium. Use a multi-channel pipette to add the mixed cells into 3 96-well plates, adding 150 μL per well. After placing them in the incubator, observe under the microscope every day, find the wells with single cells, and mark the cell division and proliferation status.

[0055] When using a conventional single-cell basal medium such as VegaCHO, the single-cell amplification efficiency is relatively low. Since single cells or a small number of cells are not easy to survive and reproduce, and in addition, the cells with some difficult-to-transfect sequences have low cell viability and weak proliferation ability. In the present invention, the proliferation activity of the LA-C1INH sequence transfected into suspension cells is significantly weaker than that of the wild-type sequence, and it is technically difficult to amplify these single cells.

[0056] In this example, recombinant human serum albumin is creatively used, which can effectively support the growth of low-density cells. Culturing low-density cells with a medium containing recombinant human serum albumin can successfully resuscitate and amplify the cells. It also avoids a series of regulatory problems brought about by using animal-derived components.

[0057] To address the problem of difficult single-cell amplification of LA-C1INH, different concentrations of recombinant human serum albumin were screened and added to the single-cell basal medium. Suspension single-cell amplification experiments were carried out according to the addition groups of (phosphate buffer solution, PBS) and recombinant human serum albumin addition groups (1 g / L, 2 g / L, 3 g / L). After two weeks of amplification culture, count the number of wells with successful final amplification in the well plates with different concentrations of albumin added (cell confluence greater than or equal to 2 / 3 volume is considered successful amplification). Use a microscope to detect and record the cell proliferation of each experimental group. Each experimental group has three parallel replicates, and the results are averaged. Finally, calculate the single-cell amplification efficiency. The results are shown in Table 1:

[0058] Table 1 Effects of different concentrations of recombinant human serum albumin on CHO single-cell amplification

[0059]

[0060] From the above data, it can be seen that the number of wells with successful cell amplification in the three experimental groups adding recombinant human serum albumin is significantly higher than that of the PBS control group. Among them, in the experimental group adding 2 g / L recombinant human serum albumin, the single-cell amplification success rate of LA-C1INH is the highest. It shows that adding 1 - 3 g / L recombinant human serum albumin can more effectively promote the amplification of suspension single cells, and preferably adding 1.5 - 2.5 g / L. The optimal concentration of recombinant human serum albumin is 2 g / L.

[0061] (3)Change the medium in the wells for single cells every day. When the cells reach 2 / 3 confluence, passage the cells to a larger well plate, and so on. When a sufficient number of single cells are amplified, take a part of them to extract genomic DNA for PCR, and sequence to identify whether the target sequence is integrated into the host cells.

[0062] PCR verification primers: Check-F: SEQ ID NO.6; Check-R: SEQ ID NO.7.

[0063] Example 3 Identification and Fed-batch Culture of Recombinant Variants of C1 Esterase Inhibitor

[0064] 1. Select the supernatants of empty vector, WT, and LA-C1INH cells for sampling. After adding SDS loading buffer, identify the size and specificity of the expressed C1-INH samples by Western blot. The results are as Figure 4 shown. Both the 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 reduced by half compared to WT.

[0065] 2. The single-cell LA-C1INH high-expression cell line obtained by amplification using the limited dilution method was cultured in a 1 L shake flask for 16 days by Fed-batch culture, and then the culture supernatant was collected. The initial culture was inoculated into vegaCHO medium (OPM, Cat.P121662) at a density of 1.0 x 10 6 cells / mL and cultured in a 37°C shaker (humidity 80%, rotation speed 130 rpm, 5% CO 2 ₂). Starting from the 2nd / 4th / 6th / 8th / 10th / 12th / 14th day, different feeds were supplemented to the culture 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 the harvest on the 16th day. On the fourth day of culture, the temperature was lowered to extend the maintenance time of the density peak. Glucose was added to the culture to maintain a culture concentration of 3 - 6 g / L. The culture medium supernatant was collected, centrifuged (2000g, 10 minutes), and stored at -20°C for subsequent analysis.

[0066] 3. To maximize the expression level of LA-C1INH, the cooling culture temperature affecting the yield of the CHO cell Fed-batch process was screened and optimized. Uniformly according to 1 x 10 6Cells were inoculated in a shake flask at a density of cells / ml. After culturing for 4 days, the culture temperature was decreased to five gradients of 31°C, 32°C, 33°C, 34°C, and 35°C from 37°C respectively, and the culture was continued until day 16. Samples were collected every day during this period to detect and record the viable cell density, cell viability, osmotic pressure, and protein expression level. The results are as Figure 5 shown. After culturing for 4 days, the temperature was decreased to 34°C. After culturing until day 16, the expression level of LA-C1INH reached the highest, and the cell peak density was maintained for a relatively long time, achieving the best expression effect.

[0067] Example 4 Purification of the recombinant variant of C1 esterase inhibitor and determination of the supernatant expression level and activity

[0068] 1. After centrifuging the collected cell supernatant at 1000 rpm at 4°C, the supernatant was filtered through a 0.22 μm filter membrane to remove impurities. The clarified treatment solution was concentrated 5 - 10 times through a 10KD membrane package. Finally, it was diafiltered with 20 mM Tris buffer. The clarified and diafiltered protein was subjected to affinity chromatography using Ni-excel packing material. The loading buffer contained 20 mM Tris, and it was washed with a 20 - 100 mM imidazole concentration gradient to elute the LA-C1INH protein, obtaining a purified LA-C1INH sample. In addition, to further improve the purity of LA-C1INH, anion chromatography was performed using a Capto Q column. Some exploration was carried out on the optimal elution conditions, and finally it was determined that the optimal elution pH for LA-C1INH was pH 8.0 (20 mM Tris, 100 mM). By detecting the protein recovery rate and purity corresponding to the elution solutions at different pH gradients (as shown in Table 2), an LA-C1INH protein with a purity close to 97% could be obtained using the pH 8.0 elution solution (the results are as Figure 6 ) and a relatively high recovery rate could be ensured.

[0069] Table 2 Statistical details of protein recovery rate and purity in the chromatography process

[0070]

[0071] 2. Expression level of LA-C1INH protein

[0072] The protein concentration of LA-C1INH in the cell supernatant was measured using an ELISA kit (abcam, ab224883) according to the instructions of the manual, and the expression level of LA-C1INH in the recombinant host cells was calculated.

[0073] The results are as Figure 7 shown. Compared with WT, the expression level of LA-C1INH protein was significantly increased, and the expression amount could reach 600 mg / L.

[0074] 3. Activity determination of LA-C1INH protein

[0075] The activity of LA-C1INH in the cell supernatant was determined using a STAGO fully automatic coagulation analyzer with reference to the method described in the article "Establishment and verification of a dynamic chromogenic detection method for human C1 esterase inhibitor activity" published in the Chinese Journal of Biologicals. The results are shown in Figure 8 .

[0076] The results showed that the activity of the purified sample of the LA-C1INH recombinant variant was significantly increased compared with that of the WT, and it had good activity.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within 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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