CN115478055A - Method for regulating hydroxylation level of recombinant protein - Google Patents

Method for regulating hydroxylation level of recombinant protein Download PDF

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CN115478055A
CN115478055A CN202110660695.6A CN202110660695A CN115478055A CN 115478055 A CN115478055 A CN 115478055A CN 202110660695 A CN202110660695 A CN 202110660695A CN 115478055 A CN115478055 A CN 115478055A
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protein
sirna
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plod2
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李京浩
梁千惠
刘阳
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Cantonbio Co ltd
Foshan Pu Jin Bioisystech Co ltd
Foshan Hanteng Biotechnology Co ltd
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Cantonbio Co ltd
Foshan Pu Jin Bioisystech Co ltd
Foshan Hanteng Biotechnology Co ltd
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Priority to PCT/CN2021/138652 priority patent/WO2022262228A1/en
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Abstract

The invention relates to a method for regulating the hydroxylation level of a recombinant protein, which comprises the following steps: reducing the expression or function of the PLOD protein in a cell expressing the recombinant protein; wherein the recombinant protein is not the PLOD protein. The method provided by the invention can adjust the hydroxylation level of the recombinant protein, thereby meeting the requirement of hydroxylation modification and adjustment in the development of biological similar drugs.

Description

Method for regulating hydroxylation level of recombinant protein
Technical Field
The invention relates to the field of molecular biology, in particular to a method for regulating the hydroxylation level of a recombinant protein.
Background
As host cells, mammals are commonly used for industrial production of recombinant proteins, and CHO cells (Chinese hamster ovary cells) are the most commonly used mammalian expression systems in industry, and are commonly used for expression and production of recombinant antibodies and recombinant proteins. Although CHO cells have been successfully used as a manufacturing host cell system for over 30 years, these cell lines are still somewhat limited in terms of growth rate and recombinant protein production capacity. Improving the performance of host cells and increasing the expression level of recombinant proteins of the host cells are always important points of attention in the field of recombinant protein production.
In recent years, the development of biosimilar drugs has been accelerated, and the level of posttranslational modification of biosimilar drugs is consistent with that of the original drug. The adjustment of post-translational modification is a difficult point in the development of biological similar drugs, and the adjustment is generally carried out by optimizing a fermentation process in industry, wherein the optimization comprises culture medium and supplementary material screening, culture parameters and the like, but the workload is large and the expected effect can not be achieved, and the hydroxylation modification proportion is a post-translational modification which is difficult to adjust through the culture process.
At present, there is a need to develop a method capable of modulating the hydroxylation level of recombinant proteins to meet the needs of hydroxylation modification modulation in the development of biologically similar drugs.
Disclosure of Invention
The object of the present invention is to overcome the drawbacks of the prior art and to provide a method for modulating the level of hydroxylation of recombinant proteins.
The invention adopts the following specific technical scheme:
in a first aspect, the present invention provides a method of modulating the level of hydroxylation of a recombinant protein, the method comprising the steps of: reducing the expression or function of the PLOD protein in a cell expressing the recombinant protein; wherein the recombinant protein is not the PLOD protein.
The method provided by the invention can reduce the hydroxylation proportion of the recombinant protein by reducing the expression or function of the PLOD protein in the cell, thereby realizing the regulation of the hydroxylation level of the recombinant protein.
In some embodiments, the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD3. Specifically, the PLOD protein is PLOD1, or PLOD2, or PLOD3, or PLOD1 and PLOD2, or PLOD1 and PLOD3, or PLOD2 and PLOD3, or PLOD1, PLOD2 and PLOD3.
In some embodiments, the PLOD protein comprises at least PLOD2.
In some embodiments, the methods administer an inhibitor that interferes with the expression or function of a PLOD protein into a cell that expresses a recombinant protein. The inhibitor can be selected from siRNA, shRNA, microRNA, antisense nucleotide, ribozyme, nucleotide or expression vector for encoding negative mutant, antibody, peptide and small molecule compound.
In some embodiments, the inhibitor comprises an siRNA directed to any one, two or three of PLOD1, PLOD2 and PLOD3. In particular, the inhibitor comprises an siRNA against PLOD1, or comprises an siRNA against PLOD2, or comprises an siRNA against PLOD3, or comprises an siRNA against PLOD1 and PLOD2, or comprises an siRNA against PLOD1 and PLOD3, or comprises an siRNA against PLOD2 and PLOD3, or comprises an siRNA against PLOD1, PLOD2 and PLOD3.
In some embodiments, the inhibitor comprises at least an siRNA against PLOD2.
In some embodiments, the siRNA against PLOD1 has a sense strand as shown in SEQ ID No.01 and an antisense strand as shown in SEQ ID No. 02.
In some embodiments, the siRNA to PLOD2 has a sense strand as shown in SEQ ID No.03 and an antisense strand as shown in SEQ ID No.04 and/or the siRNA to PLOD2 has a sense strand as shown in SEQ ID No.03 and an antisense strand as shown in SEQ ID No.04, a sense strand as shown in SEQ ID No.05 and an antisense strand as shown in SEQ ID No.06 or a sense strand as shown in SEQ ID No.07 and an antisense strand as shown in SEQ ID No. 08.
In some embodiments, the siRNA against PLOD3 has a sense strand as shown in SEQ ID No.09 and an antisense strand as shown in SEQ ID No. 10.
In some embodiments, the cell is a mammalian cell, and may be selected from CHO cells (chinese hamster ovary cells), HEK293 cells, vero cells, and the like.
In some embodiments, the cell is a CHO cell, and specifically, a cell line selected from CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, and the like can be used.
In some embodiments, the cell is a monoclonal cell expressing a recombinant protein.
In some embodiments, the cell is a cell that is exogenously transformed with a recombinant protein expression vector.
In some embodiments, the recombinant protein is a monoclonal antibody.
In some embodiments, the recombinant protein is a fusion protein. The fusion protein may be an Fc fusion protein, i.e., a protein produced by fusing a functional protein molecule having biological activity with an Fc fragment of immunoglobulin (IgG, igA, etc.) by using a technique such as genetic engineering, such as TNFR-Fc fusion protein, dolabrin, etc.
In a second aspect, the invention provides an inhibitor of the expression or function of a PLOD protein, said inhibitor comprising an siRNA to any one, two or three of PLOD1, PLOD2 and PLOD3.
In particular, the inhibitor comprises an siRNA against PLOD1, or comprises an siRNA against PLOD2, or comprises an siRNA against PLOD3, or comprises an siRNA against PLOD1 and PLOD2, or comprises an siRNA against PLOD1 and PLOD3, or comprises an siRNA against PLOD2 and PLOD3, or comprises an siRNA against PLOD1, PLOD2 and PLOD3.
The inhibitor provided by the invention can effectively inhibit the expression of PLOD protein in cells, thereby reducing the hydroxylation level of recombinant protein.
In some embodiments, the inhibitor comprises at least an siRNA against PLOD2.
In some embodiments, the siRNA against PLOD1 has a sense strand as shown in SEQ ID No.01 and an antisense strand as shown in SEQ ID No. 02.
In some embodiments, the siRNA against PLOD2 has a sense strand as shown in SEQ ID No.03 and an antisense strand as shown in SEQ ID No.04, has a sense strand as shown in SEQ ID No.05 and an antisense strand as shown in SEQ ID No.06, or has a sense strand as shown in SEQ ID No.07 and an antisense strand as shown in SEQ ID No. 08.
In some embodiments, the siRNA against PLOD3 has a sense strand as shown in SEQ ID No.09 and an antisense strand as shown in SEQ ID No. 10.
In a third aspect, the present invention provides a cell expressing a recombinant protein in which the expression or function of a PLOD protein is inhibited; wherein the recombinant protein is not the PLOD protein.
The expression or function of the PLOD protein in the cells provided by the invention is inhibited, thereby causing the hydroxylation ratio of the recombinant protein expressed by the cells to be reduced.
In some embodiments, the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD3. Specifically, the PLOD protein is PLOD1, or PLOD2, or PLOD3, or PLOD1 and PLOD2, or PLOD1 and PLOD3, or PLOD2 and PLOD3, or PLOD1, PLOD2 and PLOD3.
In some embodiments, the PLOD protein comprises at least PLOD2.
In some embodiments, the cell has exogenously added thereto an inhibitor that inhibits the expression or function of a PLOD protein. The inhibitor can be selected from siRNA, shRNA, microRNA, antisense nucleotide, ribozyme, nucleotide or expression vector for encoding negative mutant, antibody, peptide and small molecule compound.
In some embodiments, the inhibitor comprises an siRNA directed to any one, two or three of PLOD1, PLOD2 and PLOD3. In particular, the inhibitor comprises an siRNA against PLOD1, or comprises an siRNA against PLOD2, or comprises an siRNA against PLOD3, or comprises an siRNA against PLOD1 and PLOD2, or comprises an siRNA against PLOD1 and PLOD3, or comprises an siRNA against PLOD2 and PLOD3, or comprises an siRNA against PLOD1, PLOD2 and PLOD3.
In some embodiments, the inhibitor comprises at least an siRNA against PLOD2.
In some embodiments, the siRNA against PLOD1 has a sense strand as shown in SEQ ID No.01 and an antisense strand as shown in SEQ ID No. 02.
In some embodiments, the siRNA against PLOD2 has a sense strand as shown in SEQ ID No.03 and an antisense strand as shown in SEQ ID No.04, has a sense strand as shown in SEQ ID No.05 and an antisense strand as shown in SEQ ID No.06, or has a sense strand as shown in SEQ ID No.07 and an antisense strand as shown in SEQ ID No. 08.
In some embodiments, the siRNA against PLOD3 has a sense strand as shown in SEQ ID No.09 and an antisense strand as shown in SEQ ID No. 10.
In some embodiments, the cell is a mammalian cell, and may be selected from CHO cells (chinese hamster ovary cells), HEK293 cells, vero cells, and the like.
In some embodiments, the cell is a CHO cell, and specifically, a CHO-K1, CHO-S, CHO-DXB11, CHO-DG44 cell line can be selected.
In some embodiments, the cell is a monoclonal cell expressing a recombinant protein.
In some embodiments, the cell is a cell that is exogenously transformed with a recombinant protein expression vector.
In some embodiments, the recombinant protein is a monoclonal antibody.
In some embodiments, the recombinant protein is a fusion protein. The fusion protein can be Fc fusion protein, i.e., protein produced by fusing certain functional protein molecule with bioactivity with Fc fragment of immunoglobulin (IgG, igA, etc.) by using gene engineering technology, such as TNFR-Fc fusion protein, dolafetin, etc.
The siRNA protected by the invention can be an RNA sequence per se, and also comprises a form which is modified on the basis of the RNA sequence, for example, two TT bases are additionally added at the 3' end of the RNA sequence to serve as a pendulous design, so that the sequence stability is increased.
Drawings
FIG. 1 is the relative expression level of PLOD1 after 72h of siRNA transfection;
FIG. 2 is the relative expression level of PLOD2 after 72h of siRNA transfection;
FIG. 3 is the relative expression level of PLOD3 after 72h of siRNA transfection;
FIG. 4 is the relative expression level of JMJD4 at 72h after siRNA transfection;
FIG. 5 shows the viable cell density of dolastatin monoclonals 72h after siRNA transfection;
FIG. 6 shows the cell viability of dolaglutide monoclonals 72h after siRNA transfection;
FIG. 7 shows the protein yield of dolastatin monoclonals 72h after siRNA transfection;
FIG. 8 shows the ratio of the hydroxylation modification of dolastatin in the culture supernatant after 72h siRNA transfection.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Noun interpretation
siRNA: small interfering RNAs, sometimes referred to as short interfering RNAs or silencing RNAs, are a class of double-stranded RNA molecules that are 20-25 base pairs in length, resemble miRNAs, and operate within the RNA interference (RNAi) pathway. It interferes with the post-transcriptional degradation of mRNA of a particular gene expressing a nucleotide sequence complementary thereto, thereby preventing translation.
PLOD: the procollagen lysine-1,2-oxoglutarate-5-dioxygenase (PLOD) family mainly comprises three members PLOD1, PLOD2 and PLOD3 and encodes lysine hydroxylase 1 (LH 1), LH2 and LH3, respectively. The primary role of PLOD is to promote collagen maturation and secretion by catalyzing the hydroxylation of procollagen lysine residues. PLOD1, PLOD2 and PLOD3 have different substrate specificities, and each can recognize and hydroxylate lysine in different domains of the collagen propeptide.
JMJD4: the protein family (JMJD) containing Jumonji structural domains has more varieties and various catalytic substrates, and generally needs ferrous ions and alpha-ketoglutaric acid to participate; the N-terminal and C-terminal of the family members both contain a characteristic domain (called JmJN and JmJC respectively) of the transcription factor family Jumonji, wherein the JmJN domainIs related to transcriptional regulation, and JmjC is one of the components of the enzymatic activity center of the JMJD family; JMJD4 is one of the members of this family, a hydroxylase involved in post-translational modification.
Dolaglutide: dulaglutide (trade name:
Figure BDA0003115097200000061
) The long-acting GLP-1R agonist is a novel long-acting GLP-1R agonist developed by Lily company in America, is obtained by fusing two GLP-1 analogs with DPP-4 inhibitory action and a human immunoglobulin heavy chain IgG4-Fc fragment, has activity similar to that of endogenous GLP-1 and half-life of 5d, and can effectively delay the clearance action of kidney. The FDA approved dolabrus peptide subcutaneous injection for marketing in 2014 at 9 months. The european commission approved duraglutide subcutaneous injection on europe at 12 months 2014.
Example 1: siRNA design and Synthesis
This example designed multiple sets of siRNA for the sequences of PLOD1 (NCBI accession number XM _ 003514397.3), PLOD2 (NCBI accession number XM _ 035459128.1), PLOD3 (NCBI accession number XM _ 035454906.1), JMJD4 (NCBI accession number XM _ 035456468.1), respectively, and synthesized and annealed to double strands at Beijing Rui Boxing Ke Biotechnology Limited. The sequences of typical sirnas are shown in table 1 below.
Table 1: siRNA sequences
Figure BDA0003115097200000071
Figure BDA0003115097200000081
In the siRNA sequence shown in Table 1, two additional "TT" bases can be added at the 3' end of the RNA sequence itself as a pendulous design, thereby increasing the sequence stability and increasing the half-life before the formation of RICS.
In the subsequent examples of the present invention, siRNA forms with "TT" added to the 3' end were used.
Example 2: siRNA transfection
1. Experimental materials:
host cells and culture conditions: CHO-K1 monoclonal for expressing dolabrin; culture medium: EX-Cell Advanced CHO Fed-batch medium (sigma); before experiment, the monoclonal cell is revived and passaged for more than one week, and the inoculation density is 0.3-0.5 × 10 6 One/ml, passage once every 3 days, at 180rpm,5% CO 2 Culturing in a cell culture shaker;
transfection medium: hycell TransFx-C (hyclone);
transfection reagent: RNATransMate (shanghai bio).
2. The experimental steps are as follows:
monoclonal cells were passaged to 1.0X 10 the day before transfection 6 Counts on the day of transfection per ml, adjusted to a density of 2.6X 10 with transfection medium Hycell TransFx-C (hyclone) 6 And each sample is divided into 125ml shake flasks, and each flask is 19ml. The siRNA and transfection reagent RNAtrasmate with the highest knockdown efficiency in each group in Table 1 were diluted into 3ml of Hycell TransFx-C (hyclone), and the diluted siRNA and RNAtrasmate were mixed uniformly and then left to stand for 5-10 minutes. Adding into cell suspension in shake flask with final volume of 25ml, and placing back on shaking table for further culture. The total cell mass, siRNA and transfection reagent amounts are shown in table 2.
Table 2: cell, siRNA and transfection reagent dosage
Figure BDA0003115097200000091
Figure BDA0003115097200000101
Counting at 72h after transfection, centrifuging the cell suspension for 200g,10 min, using the cells for RNA extraction, performing reverse transcription after the RNA extraction is finished to obtain cDNA, and quantitatively detecting the expression quantity of each gene to confirm the knock-down effect; and (3) the supernatant is used for detecting and purifying the titer of the dolabrin, and the hydroxylation modification proportion of the dolabrin protein is detected by HPLC peptide map analysis after the purification is finished.
3. Results of the experiment
3.1 knockdown efficiency 72h after siRNA transfection:
since PLOD1, PLOD2 and PLOD3 are lysine hydroxylase, in order to find out whether knocking down one of the other two genes will increase expression in a compensatory manner, in this example, siRNA of one of PLODs is transfected separately, and then expression levels of three PLODs are detected simultaneously, JMJD4 only detects expression of itself.
From the results, it can be seen that: si-PLOD1 is transfected separately, and the expression level of PLOD1 gene is 32% of that of the control group (as shown in figure 1); si-PLOD2 was transfected alone, and the expression level of PLOD2 was 43% of that of the control group (as shown in FIG. 2); si-PLOD3 was transfected alone, with PLOD3 expression at 40% of the control (as shown in FIG. 3); the expression level of transfected si-JMJD4 and JMJD4 is 41% of that of the control group (as shown in FIG. 4), and the above results show that the siRNA achieves the knock-down effect. Moreover, in the group co-transfected with si-PLOD1, si-PLOD2 and si-PLOD3, the expression of all three genes was reduced compared with the control group, which was 46%, 35% and 42% of the expression level of the control group, respectively, and in addition, knocking down one of the PLODs had no effect on the expression levels of the other two PLODs (as shown in FIG. 1, FIG. 2 and FIG. 3).
And (3) knotting: the siRNA provided by the invention can respectively achieve the effect on the knockdown of 4 genes PLOD1, PLOD2, PLOD3 and JMJD4, and one of the three gene knockdown of PLOD has little influence on the expression of the other two genes.
3.2 Effect on cell live cell Density after knock-Down hydroxylase
As shown in fig. 5, the viable cell density of the mixture of PLOD1, PLOD2, PLOD3, (PLOD 1+ PLOD2+ PLOD 3) and JMJD4 was slightly decreased after siRNA transfection compared to the control group, which was 83%,91%,87%,92% and 83% of the control group, respectively.
3.3 Effect on cell viability after knockdown of hydroxylase
As shown in fig. 6, after knockdown of PLOD1, PLOD2, PLOD3, (PLOD 1+ PLOD2+ PLOD 3) and JMJD4 after siRNA transfection, no significant effect on cell viability was observed, indicating that knockdown hydroxylase cells can also substantially maintain normal growth.
3.4 Effect of hydroxylase knockdown on cell monoclonal yield
As shown in fig. 7, after knockdown of PLOD1, PLOD2, PLOD3, (PLOD 1+ PLOD2+ PLOD 3) and JMJD4 after siRNA transfection, there was a partial reduction in protein production, which was 69%,76%,75%,82% and 84% of the control group, respectively.
3.5 Effect of knockdown of hydroxylase on the modification ratio of hydroxylation of Doralutin
As shown in fig. 8, the knockdown PLOD2 can significantly reduce the dolabrus peptide hydroxylation modification ratio from 21% to 13% in the control group, the knockdown PLOD1 dolabrus peptide hydroxylation modification ratio is 17%, the knockdown PLOD3 and JMJD4 have no significant effect on the hydroxylation modification ratio, and the joint knockdown of PLOD1, PLOD2 and PLOD3 also reduces the hydroxylation modification ratio to 13%. The above results indicate that knocking down PLOD2 can significantly reduce the protein hydroxylation modification ratio.
And (3) knotting: in the present example, after knocking down PLOD2, the viable cell density was 91% of that of the control group, and the protein production was 76% of that of the control group, but the cell viability rate was not significantly different from that of the control group. In general, the method provided by the invention for knocking down PLOD2 has little influence on cell growth, survival and protein yield, but can obviously reduce the hydroxylation modification proportion of the recombinant protein, and can be used for hydroxylation modification regulation in the development of biological similar drugs.
It will be appreciated by those skilled in the art that, in addition to the siRNA techniques used in the examples of the invention, the reduction of expression or function of PLOD proteins in cells in the invention may also be achieved by shRNA techniques or any gene editing technique known in the art. Exemplary gene editing techniques include regular clustered, interspersed short palindromic repeats (CRISPR), zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) techniques.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
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CANTONBIO Co.,Ltd.
Foshan Pu Jin Bioisystech Co.,Ltd.
<120> a method for modulating the level of hydroxylation of a recombinant protein
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acgccaagcu ugaggacaa 19
<210> 2
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
uuguccucaa gcuuggcgu 19
<210> 3
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccgaauaucu ggugguuau 19
<210> 4
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
auaaccacca gauauucgg 19
<210> 5
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcgcaucccu gcagauaaa 19
<210> 6
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
uuuaucugca gggaugcgc 19
<210> 7
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcagucgaug uucauccaa 19
<210> 8
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
uuggaugaac aucgacugc 19
<210> 9
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcuaugacac ugaccacuu 19
<210> 10
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aaguggucag ugucauagc 19
<210> 11
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggaggugaug uugcucgaa 19
<210> 12
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
uucgagcaac aucaccucc 19
<210> 13
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gcccagaugu guacugguu 19
<210> 14
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aaccaguaca caucugggc 19
<210> 15
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ccaucucuau caaccacaa 19
<210> 16
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
uugugguuga uagagaugg 19
<210> 17
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ccuuccaugc ggacaucuu 19
<210> 18
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aagauguccg cauggaagg 19
<210> 19
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggaagaaaug guuguucuu 19
<210> 20
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aagaacaacc auuucuucc 19
<210> 21
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cuacguccag gagcgcacc 19
<210> 22
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ggugcgcucc uggacguag 19

Claims (10)

1. A method of modulating the level of hydroxylation of a recombinant protein comprising the steps of: reducing the expression or function of the PLOD protein in a cell expressing the recombinant protein; wherein the recombinant protein is not the PLOD protein.
2. The method of claim 1, wherein the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD 3;
preferably, the PLOD protein comprises at least PLOD2.
3. The method of claim 1, wherein the method comprises administering an inhibitor that interferes with the expression or function of the PLOD protein into a cell expressing the recombinant protein; the inhibitor is selected from siRNA, shRNA, microRNA, antisense nucleotide, ribozyme, nucleotide or expression vector for coding negative mutant, antibody, peptide and small molecular compound;
preferably, the inhibitor comprises an siRNA directed to any one, two or three of PLOD1, PLOD2 and PLOD3, preferably at least an siRNA directed to PLOD2.
4. The method of claim 3, wherein the siRNA to PLOD1 has a sense strand as set forth in SEQ ID No.01 and an antisense strand as set forth in SEQ ID No. 02;
and/or, the siRNA aiming at PLOD2 has a sense strand shown as SEQ ID NO.03 and an antisense strand shown as SEQ ID NO.04, has a sense strand shown as SEQ ID NO.05 and an antisense strand shown as SEQ ID NO.06, or has a sense strand shown as SEQ ID NO.07 and an antisense strand shown as SEQ ID NO. 08;
and/or, the siRNA aiming at PLOD3 has a sense strand shown as SEQ ID NO.09 and an antisense strand shown as SEQ ID NO. 10.
5. The method according to any one of claims 1 to 4, wherein the cells are mammalian cells, preferably selected from the group consisting of CHO cells, HEK293 cells and Vero cells;
preferably, the cell is a monoclonal cell for expressing the recombinant protein or a cell exogenously transferred with a recombinant protein expression vector; the recombinant protein is preferably selected from the group consisting of monoclonal antibodies and fusion proteins, the fusion protein further preferably being an Fc fusion protein.
6. An inhibitor that interferes with the expression or function of a PLOD protein, wherein said inhibitor comprises an siRNA directed to any one, two or three of PLOD1, PLOD2 and PLOD3, preferably at least an siRNA directed to PLOD 2;
preferably, the siRNA to PLOD1 has a sense strand as shown in SEQ ID NO.01 and an antisense strand as shown in SEQ ID NO. 02; and/or, the siRNA aiming at PLOD2 has a sense strand shown as SEQ ID NO.03 and an antisense strand shown as SEQ ID NO.04, has a sense strand shown as SEQ ID NO.05 and an antisense strand shown as SEQ ID NO.06, or has a sense strand shown as SEQ ID NO.07 and an antisense strand shown as SEQ ID NO. 08; and/or, the siRNA aiming at PLOD3 has a sense strand shown as SEQ ID NO.09 and an antisense strand shown as SEQ ID NO. 10.
7. A cell expressing a recombinant protein, wherein the expression or function of a PLOD protein in said cell is inhibited; wherein the recombinant protein is not the PLOD protein;
preferably, the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD3, preferably including at least PLOD2.
8. The cell of claim 7, wherein an inhibitor that inhibits the expression or function of a PLOD protein is exogenously added to the cell, wherein the inhibitor is selected from the group consisting of siRNA, shRNA, microRNA, antisense nucleotides, ribozymes, nucleotides or expression vectors encoding negative mutants, antibodies, peptides and small molecule compounds;
preferably, the inhibitor comprises an siRNA against any one, two or three of PLOD1, PLOD2 and PLOD3, preferably at least an siRNA against PLOD2.
9. The cell of claim 8, wherein the siRNA to PLOD1 has a sense strand as set forth in SEQ ID No.01 and an antisense strand as set forth in SEQ ID No. 02;
and/or, the siRNA aiming at PLOD2 has a sense strand shown as SEQ ID NO.03 and an antisense strand shown as SEQ ID NO.04, has a sense strand shown as SEQ ID NO.05 and an antisense strand shown as SEQ ID NO.06, or has a sense strand shown as SEQ ID NO.07 and an antisense strand shown as SEQ ID NO. 08;
and/or, the siRNA aiming at PLOD3 has a sense strand shown as SEQ ID NO.09 and an antisense strand shown as SEQ ID NO. 10.
10. The cell according to any one of claims 7 to 9, wherein the cell is a mammalian cell, preferably selected from the group consisting of CHO cells, HEK293 cells and Vero cells;
preferably, the cell is a monoclonal cell for expressing the recombinant protein or a cell which is exogenously transferred with a recombinant protein expression vector; the recombinant protein is preferably selected from the group consisting of monoclonal antibodies and fusion proteins, the fusion protein further preferably being an Fc fusion protein.
CN202110660695.6A 2021-06-15 2021-06-15 Method for regulating hydroxylation level of recombinant protein Pending CN115478055A (en)

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