WO2022262228A1 - 一种调节重组蛋白羟基化水平的方法 - Google Patents
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Images
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/10—Cells modified by introduction of foreign genetic material
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the invention relates to the field of molecular biology, in particular to a method for regulating the hydroxylation level of recombinant proteins.
- CHO cells Choinese hamster ovary cells
- CHO cells have been successfully used as a manufacturing host cell system for more than 30 years, these cell lines are still limited in terms of growth rate and recombinant protein production capacity. Improving the performance of host cells and increasing the expression of recombinant proteins in host cells have always been the focus of attention in the field of recombinant protein production.
- the purpose of the present invention is to overcome the defects of the prior art and provide a method for regulating the hydroxylation level of recombinant protein.
- the present invention adopts following concrete technical scheme:
- the present invention provides a method for regulating the hydroxylation level of a recombinant protein, the method comprising the steps of: reducing the expression or function of the PLOD protein in cells expressing the recombinant protein; wherein the recombinant protein is not the PLOD protein.
- the method provided by the invention can reduce the hydroxylation ratio of the recombinant protein by reducing the expression or function of the PLOD protein in cells, thereby realizing the regulation of the hydroxylation level of the recombinant protein.
- the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD3.
- 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.
- the PLOD protein includes at least PLOD2.
- the method administers to cells expressing the recombinant protein an inhibitor that interferes with the expression or function of the PLOD protein.
- the inhibitor can be selected from siRNA, shRNA, microRNA, antisense nucleotides, ribozymes, nucleotides encoding negative mutants or expression vectors, antibodies, peptides and small molecule compounds.
- the inhibitor comprises siRNA against any one, two, or three of PLOD1, PLOD2, and PLOD3.
- the inhibitor includes siRNA against PLOD1, or siRNA against PLOD2, or siRNA against PLOD3, or siRNA against PLOD1 and PLOD2, or siRNA against PLOD1 and PLOD3, or siRNA against PLOD2 and PLOD3, or include siRNAs against PLOD1, PLOD2, and PLOD3.
- the inhibitor comprises at least siRNA against PLOD2.
- 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.
- the siRNA against PLOD2 has a sense strand as shown in SEQ ID NO.03 and the antisense strand as shown in SEQ ID NO.04 and/or, the siRNA against PLOD2 has a strand as shown in SEQ ID NO.03
- the sense strand shown and the antisense strand as shown in SEQ ID NO.04 have the sense strand as shown in SEQ ID NO.05 and the antisense strand as shown in SEQ ID NO.06 or have the sense strand as shown in SEQ ID NO.06
- 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.
- the cells are mammalian cells, which can be selected from CHO cells (Chinese hamster ovary cells), HEK293 cells, Vero cells and the like.
- the cells are CHO cells, specifically CHO-K1, CHO-S, CHO-DXB11, CHO-DG44 and other cell lines can be selected.
- the cells are monoclonal cells expressing recombinant proteins.
- the cells are exogenously transfected with recombinant protein expression vectors.
- the recombinant protein is a monoclonal antibody.
- the recombinant protein is a fusion protein.
- the fusion protein may be an Fc fusion protein, that is, a protein produced by fusing a certain biologically active functional protein molecule with an Fc fragment of an immunoglobulin (IgG, IgA, etc.) using techniques such as genetic engineering, such as TNFR-Fc fusion protein, dulaglutide, etc.
- the present invention provides an inhibitor that interferes with the expression or function of PLOD protein, and the inhibitor includes siRNA against any one, two or three of PLOD1, PLOD2 and PLOD3.
- the inhibitor includes siRNA against PLOD1, or siRNA against PLOD2, or siRNA against PLOD3, or siRNA against PLOD1 and PLOD2, or siRNA against PLOD1 and PLOD3, or siRNA against PLOD2 and PLOD3, or include siRNAs 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.
- the inhibitor comprises at least siRNA against PLOD2.
- 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.
- 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 a sense strand as shown in SEQ ID NO.05
- 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.
- the present invention provides a cell expressing a recombinant protein, in which the expression or function of the 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 reducing the hydroxylation ratio of the recombinant protein expressed in the cells.
- the PLOD protein is selected from any one, two or three of PLOD1, PLOD2 and PLOD3.
- 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.
- the PLOD protein includes at least PLOD2.
- an inhibitor that inhibits the expression or function of the PLOD protein is added exogenously to the cells.
- the inhibitor can be selected from siRNA, shRNA, microRNA, antisense nucleotides, ribozymes, nucleotides encoding negative mutants or expression vectors, antibodies, peptides and small molecule compounds.
- the inhibitor comprises siRNA against any one, two, or three of PLOD1, PLOD2, and PLOD3.
- the inhibitor includes siRNA against PLOD1, or siRNA against PLOD2, or siRNA against PLOD3, or siRNA against PLOD1 and PLOD2, or siRNA against PLOD1 and PLOD3, or siRNA against PLOD2 and PLOD3, or include siRNAs against PLOD1, PLOD2, and PLOD3.
- the inhibitor comprises at least siRNA against PLOD2.
- 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.
- 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 a sense strand as shown in SEQ ID NO.05
- 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.
- the cells are mammalian cells, which can be selected from CHO cells (Chinese hamster ovary cells), HEK293 cells, Vero cells and the like.
- the cells are CHO cells, specifically CHO-K1, CHO-S, CHO-DXB11, CHO-DG44 and other cell lines can be selected.
- the cells are monoclonal cells expressing recombinant proteins.
- the cells are exogenously transfected with recombinant protein expression vectors.
- the recombinant protein is a monoclonal antibody.
- the recombinant protein is a fusion protein.
- the fusion protein may be an Fc fusion protein, that is, a protein produced by fusing a certain biologically active functional protein molecule with an Fc fragment of an immunoglobulin (IgG, IgA, etc.) using techniques such as genetic engineering, such as TNFR-Fc fusion protein, dulaglutide, etc.
- the siRNA protected by the present invention can be the RNA sequence itself, or a modified form based on the RNA sequence, such as adding two "TT" bases at the 3' end of the RNA sequence as an overhang design, thereby increasing the sequence stability.
- Fig. 1 is the relative expression level of PLOD1 after siRNA transfection 72h;
- Fig. 2 is the relative expression level of PLOD2 after siRNA transfection 72h;
- Fig. 3 is the relative expression level of PLOD3 after siRNA transfection 72h;
- Fig. 4 is the relative expression level of JMJD4 after siRNA transfection 72h;
- Figure 5 is the living cell density of dulaglutide monoclonal after siRNA transfection 72h;
- Figure 6 is the cell viability of dulaglutide monoclonal after siRNA transfection 72h
- Fig. 7 is the protein yield of dulaglutide monoclonal after siRNA transfection 72h;
- Figure 8 shows the hydroxylation modification ratio of dulaglutide in the culture supernatant 72 hours after siRNA transfection.
- siRNA Small interfering RNA, sometimes called short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length, similar to miRNA, and operates within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences to post-transcriptional degradation of mRNA, thereby preventing translation.
- RNAi RNA interference
- PLOD The procollagen lysine-1,2-oxoglutarate-5-dioxygenase (PLOD) family mainly includes three members, PLOD1, PLOD2 and PLOD3, which encode lysine hydroxylase 1 (LH1 ), LH2 and LH3.
- the main role of PLOD is to promote collagen maturation and secretion by catalyzing the hydroxylation of lysine residues in procollagen.
- PLOD1, PLOD2, and PLOD3 have different substrate specificities, and each can recognize and hydroxylate lysines in different domains in collagen propeptides.
- JMJD4 Jumonji domain-containing protein (JMJD) has many kinds of proteins, and its catalytic substrates are diverse, generally requiring the participation of ferrous ions and ⁇ -ketoglutarate; the N-terminal and The C-terminus contains a characteristic domain of the transcription factor family Jumonji (referred to as JmjN and JmjC), in which the JmjN domain is related to transcriptional regulation, and JmjC is one of the components of the JMJD family enzyme activity center; JMJD4 is a member of this family One, is hydroxylase involved in post-translational modification.
- Dulaglutide (trade name: ), is a new type of long-acting GLP-1R agonist developed by Lily Company in the United States. It is obtained by fusion of two GLP-1 analogues with DPP-4 inhibitory effect and human immunoglobulin heavy chain IgG4-Fc fragment. Its activity is similar to that of Endogenous GLP-1 is similar, with a half-life of 5 days, which can effectively delay the clearance of the kidney.
- Example 1 siRNA design and synthesis
- multiple groups are designed for the sequences of PLOD1 (NCBI acquisition number XM_003514397.3), PLOD2 (NCBI acquisition number XM_035459128.1), PLOD3 (NCBI acquisition number XM_035454906.1), JMJD4 (NCBI acquisition number XM_035456468.1) siRNA was synthesized and annealed into double strands at Beijing Ruibo Xingke Biotechnology Co., Ltd. The sequences of typical siRNAs are shown in Table 1 below.
- siRNA sequence shown in Table 1 two additional "TT" bases can be added at the 3' end of the RNA sequence itself as an overhang design, thereby increasing sequence stability and increasing its half-life before the formation of RICS.
- siRNA form with "TT" added to the 3' end is used.
- Host cells and culture conditions CHO-K1 monoclonal expressing dulaglutide; medium: EX-Cell Advanced CHO Fed-batch medium (sigma); the monoclonal cells have been recovered and passaged for more than one week before the experiment, and the seeding density is 0.3-0.5 ⁇ 10 6 cells/ml, subculture once every 3 days, and culture in 180rpm, 5% CO 2 cell culture shaker;
- Transfection medium Hycell TransFx-C (hyclone);
- RNATransMate (Shanghai Sangong).
- RNA extraction After transfection, count at 72h, centrifuge the cell suspension at 200g for 10 minutes, and use the cells for RNA extraction. After the RNA extraction is completed, it is reverse transcribed into cDNA, and the expression of each gene is quantitatively detected to confirm the knockdown effect; the supernatant is used for Du Laglutide titer detection and purification, after the purification is completed, the hydroxylation modification ratio of dulaglutide protein is detected by HPLC peptide map analysis.
- PLOD1, PLOD2, and PLOD3 are all lysine hydroxylases, in order to find out whether knocking down one of the other two genes will compensatory increase the expression, in this example, after transfecting one of the siRNAs of PLOD, PLOD three The expression level of each gene is detected, while JMJD4 only detects its own expression.
- si-PLOD1 was transfected alone, and the expression level of PLOD1 gene was 32% of the control group (as shown in Figure 1); while si-PLOD2 was transfected alone, the expression level of PLOD2 was 43% of the control group (as shown in Figure 2 shown); alone transfected with si-PLOD3, the expression level of PLOD3 was 40% of the control group (as shown in Figure 3); transfected with si-JMJD4, the expression level of JMJD4 was 41% of the control group (as shown in Figure 4) , the above results show that siRNA achieves a knockdown effect.
- si-PLOD1 si-PLOD2 and si-PLOD3 co-transfection groups
- the expressions of the three genes were all decreased compared with the control group, which were 46%, 35% and 42% of the expression levels of the control group respectively.
- knockout Reducing one of the PLODs has no effect on the expression of the other two PLODs (as shown in Figure 1, Figure 2 and Figure 3).
- the siRNAs provided by the present invention can respectively achieve knockdown effects on the four genes PLOD1, PLOD2, PLOD3, and JMJD4, and the knockdown of one of the three PLOD genes has little effect on the expression of the other two.
- knocking down PLOD2 can significantly reduce the hydroxylation modification ratio of dulaglutide from 21% in the control group to 13%
- knocking down PLOD1 dulaglutide hydroxylation modification ratio is 17%
- knocking down PLOD3 PLOD1, PLOD2, and PLOD3 co-knockdown also reduced the hydroxylation modification ratio to 13%.
- the above results show that knocking down PLOD2 can significantly reduce the proportion of protein hydroxylation modification.
- knocking down PLOD2 after knocking down PLOD2, the viable cell density was 91% of the control group, and the protein production was 76% of the control group, but there was no significant difference in cell viability compared with the control group.
- knocking down PLOD2 with the method provided by the present invention has little effect on cell growth, survival and protein production, but can significantly reduce the proportion of hydroxylation modification of recombinant proteins, which can be used for regulation of hydroxylation modification in the development of biosimilar drugs.
- reducing the expression or function of PLOD protein in cells in the present invention can also be achieved by shRNA technology or any gene editing technology known in the art.
- exemplary gene editing techniques include regular clustering of short palindrome repeats (CRISPR), zinc finger nuclease (ZFN), transcription activator-like nuclease (transcription activator-like) effector nuclease, TALEN) technology.
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Abstract
提供一种调节重组蛋白羟基化水平的方法,包括如下步骤:在表达重组蛋白的细胞中,降低PLOD蛋白的表达或功能;该重组蛋白不是PLOD蛋白。该方法可以调节重组蛋白羟基化水平,从而满足生物类似药开发中的羟基化修饰调节的需要。
Description
本发明涉及分子生物学领域,具体涉及一种调节重组蛋白羟基化水平的方法。
重组蛋白工业生产的常用哺乳动物作为宿主细胞,CHO细胞(中国仓鼠卵巢细胞)是工业上最常用的哺乳动物表达系统,常用于重组抗体及重组蛋白的表达和生产。尽管CHO细胞已成功地用作制造宿主细胞系统超过30年,但这些细胞系在生长速度和重组蛋白生产能力方面仍然受到一定限制。改善宿主细胞的性能,提高宿主细胞的重组蛋白表达量,一直都是重组蛋白生产领域关注的重点。
近年来,生物类似药的发展迅速,生物类似药的翻译后修饰水平与原研药一致是其中一个重要的指标。调节翻译后修饰是生物类似药开发中的难点,工业上一般采用发酵工艺的优化来调节,包括培养基及补料筛选,培养参数等,但工作量大且不一定能达到预期效果,其中,羟基化修饰比例更是通过培养工艺难以调节的一种翻译后修饰。
目前,亟待开发一种能够调节重组蛋白羟基化水平的方法,以满足生物类似药开发中的羟基化修饰调节的需要。
发明内容
本发明的目的在于克服现有技术的缺陷,提供一种调节重组蛋白羟基化水平的方法。
本发明采用如下具体技术方案:
第一方面,本发明提供一种调节重组蛋白羟基化水平的方法,该方法包括如下步骤:在表达重组蛋白的细胞中,降低PLOD蛋白的表 达或功能;其中,所述重组蛋白不为所述PLOD蛋白。
本发明提供的方法通过降低细胞中PLOD蛋白的表达或功能,可以降低重组蛋白的羟基化比例,从而实现对重组蛋白羟基化水平的调节。
在一些实施方式中,所述PLOD蛋白选自PLOD1、PLOD2和PLOD3中的任意一种、两种或三种。具体而言,所述PLOD蛋白为PLOD1,或为PLOD2,或为PLOD3,或为PLOD1和PLOD2,或为PLOD1和PLOD3,或为PLOD2和PLOD3,或为PLOD1、PLOD2和PLOD3。
在一些实施方式中,所述PLOD蛋白至少包括PLOD2。
在一些实施方式中,所述方法将干扰PLOD蛋白的表达或功能的抑制剂施用于表达重组蛋白的细胞中。所述抑制剂可以选自siRNA、shRNA、microRNA、反义核苷酸、核酶、编码负性突变体的核苷酸或表达载体、抗体、肽和小分子化合物。
在一些实施方式中,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA。具体而言,所述抑制剂包括针对PLOD1的siRNA,或包括针对PLOD2的siRNA,或包括针对PLOD3的siRNA,或包括针对PLOD1和PLOD2的siRNA,或包括针对PLOD1和PLOD3的siRNA,或包括针对PLOD2和PLOD3的siRNA,或包括针对PLOD1、PLOD2和PLOD3的siRNA。
在一些实施方式中,所述抑制剂至少包括针对PLOD2的siRNA。
在一些实施方式中,针对PLOD1的siRNA具有如SEQ ID NO.01所示的正义链和如SEQ ID NO.02所示的反义链。
在一些实施方式中,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链和/或,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08 所示的反义链。
在一些实施方式中,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
在一些实施方式中,所述细胞为哺乳动物细胞,可以选自CHO细胞(中国仓鼠卵巢细胞)、HEK293细胞、Vero细胞等。
在一些实施方式中,所述细胞为CHO细胞,具体可以选用CHO-K1、CHO-S、CHO-DXB11、CHO-DG44等细胞系。
在一些实施方式中,所述细胞为表达重组蛋白的单克隆细胞。
在一些实施方式中,所述细胞为外源转入了重组蛋白表达载体的细胞。
在一些实施方式中,所述重组蛋白为单克隆抗体。
在一些实施方式中,所述重组蛋白为融合蛋白。所述融合蛋白可以是Fc融合蛋白,即利用基因工程等技术将某种具有生物活性的功能蛋白分子与免疫球蛋白(IgG、IgA等)的Fc片段融合而产生的蛋白,如TNFR-Fc融合蛋白、杜拉鲁肽等。
第二方面,本发明提供一种干扰PLOD蛋白的表达或功能的抑制剂,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA。
具体而言,所述抑制剂包括针对PLOD1的siRNA,或包括针对PLOD2的siRNA,或包括针对PLOD3的siRNA,或包括针对PLOD1和PLOD2的siRNA,或包括针对PLOD1和PLOD3的siRNA,或包括针对PLOD2和PLOD3的siRNA,或包括针对PLOD1、PLOD2和PLOD3的siRNA。
本发明提供的抑制剂可以有效抑制细胞中PLOD蛋白的表达,从而降低重组蛋白的羟基化水平。
在一些实施方式中,所述抑制剂至少包括针对PLOD2的siRNA。
在一些实施方式中,针对PLOD1的siRNA具有如SEQ ID NO.01 所示的正义链和如SEQ ID NO.02所示的反义链。
在一些实施方式中,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08所示的反义链。
在一些实施方式中,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
第三方面,本发明提供一种表达重组蛋白的细胞,所述细胞中PLOD蛋白的表达或功能被抑制;其中,所述重组蛋白不为所述PLOD蛋白。
本发明提供的细胞中PLOD蛋白的表达或功能被抑制,从而导致细胞表达得到的重组蛋白的羟基化比例降低。
在一些实施方式中,所述PLOD蛋白选自PLOD1、PLOD2和PLOD3中的任意一种、两种或三种。具体而言,所述PLOD蛋白为PLOD1,或为PLOD2,或为PLOD3,或为PLOD1和PLOD2,或为PLOD1和PLOD3,或为PLOD2和PLOD3,或为PLOD1、PLOD2和PLOD3。
在一些实施方式中,所述PLOD蛋白至少包括PLOD2。
在一些实施方式中,所述细胞中外源加入了抑制PLOD蛋白的表达或功能的抑制剂。所述抑制剂可以选自siRNA、shRNA、microRNA、反义核苷酸、核酶、编码负性突变体的核苷酸或表达载体、抗体、肽和小分子化合物。
在一些实施方式中,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA。具体而言,所述抑制剂包括针对PLOD1的siRNA,或包括针对PLOD2的siRNA,或包括针对PLOD3的siRNA,或包括针对PLOD1和PLOD2的siRNA,或包括针对PLOD1和PLOD3的siRNA,或包括针对PLOD2和PLOD3的siRNA,或包括针对PLOD1、PLOD2和PLOD3的siRNA。
在一些实施方式中,所述抑制剂至少包括针对PLOD2的siRNA。
在一些实施方式中,针对PLOD1的siRNA具有如SEQ ID NO.01所示的正义链和如SEQ ID NO.02所示的反义链。
在一些实施方式中,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08所示的反义链。
在一些实施方式中,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
在一些实施方式中,所述细胞为哺乳动物细胞,可以选自CHO细胞(中国仓鼠卵巢细胞)、HEK293细胞、Vero细胞等。
在一些实施方式中,所述细胞为CHO细胞,具体可以选用CHO-K1、CHO-S、CHO-DXB11、CHO-DG44等细胞系。
在一些实施方式中,所述细胞为表达重组蛋白的单克隆细胞。
在一些实施方式中,所述细胞为外源转入了重组蛋白表达载体的细胞。
在一些实施方式中,所述重组蛋白为单克隆抗体。
在一些实施方式中,所述重组蛋白为融合蛋白。所述融合蛋白可以是Fc融合蛋白,即利用基因工程等技术将某种具有生物活性的功能蛋白分子与免疫球蛋白(IgG、IgA等)的Fc片段融合而产生的蛋白,如TNFR-Fc融合蛋白、杜拉鲁肽等。
本发明所保护的siRNA可以是RNA序列本身,也包括在RNA序列的基础上经过一定修饰的形式,例如在RNA序列的3’端额外增加两个“TT”碱基作为悬垂设计,从而增加序列稳定性。
图1为siRNA转染72h后PLOD1的相对表达水平;
图2为siRNA转染72h后PLOD2的相对表达水平;
图3为siRNA转染72h后PLOD3的相对表达水平;
图4为siRNA转染72h后JMJD4的相对表达水平;
图5为siRNA转染72h后杜拉鲁肽单克隆的活细胞密度;
图6为siRNA转染72h后杜拉鲁肽单克隆的细胞活率;
图7为siRNA转染72h后杜拉鲁肽单克隆的蛋白产量;
图8为siRNA转染72h后培养上清中杜拉鲁肽羟基化修饰比例。
以下实施例用于说明本发明,但不用来限制本发明的范围。
名词解释
siRNA:小干扰RNA,有时称为短干扰RNA或沉默RNA,是一类双链RNA分子,长度为20-25个碱基对,类似于miRNA,并且在RNA干扰(RNAi)途径内操作。它干扰了表达与互补的核苷酸序列的特定基因的转录后降解的mRNA,从而防止翻译。
PLOD:前胶原赖氨酸-1,2-酮戊二酸-5-双加氧酶(PLOD)家族主要包括三个成员PLOD1,PLOD2和PLOD3,并分别编码赖氨酸羟化酶1(LH1),LH2和LH3。PLOD的主要作用是通过催化原胶原赖氨酸残基的羟基化来促进胶原蛋白的成熟和分泌。PLOD1,PLOD2和PLOD3具有不同的底物特异性,各自可以识别胶原蛋白前肽中不同结构域的赖氨酸并将其羟基化。
JMJD4:包含Jumonji结构域的蛋白质家族(Jumonji domain-containing protein,JMJD)种类较多,催化底物多样,一般需要二价铁离子和α-酮戊二酸的参与;该家族成员的N端和C端都包含一个转录因子家族Jumonji的特征结构域(分别称为JmjN和JmjC),其中JmjN结构域与转录调节相关,而JmjC是JMJD家族酶活性中心的组成之一;JMJD4是该家族的成员之一,是参与到翻译后修饰的羟化酶。
杜拉鲁肽:Dulaglutide(商品名:
),是由美国Lily公 司研发的新型长效GLP-1R激动药,由两个具有DPP-4抑制作用的GLP-1类似物和人免疫球蛋白重链IgG4-Fc片段融合得到,其活性与内源性GLP-1相似,半衰期为5d,能有效延缓肾脏的清除作用。FDA于2014年9月批准杜拉鲁肽皮下注射液上市。欧盟委员会于2014年12月批准杜拉鲁肽皮下注射液在欧洲上市。
实施例1:siRNA设计与合成
本实施例针对PLOD1(NCBI获得号XM_003514397.3),PLOD2(NCBI获得号XM_035459128.1),PLOD3(NCBI获得号XM_035454906.1),JMJD4(NCBI获得号XM_035456468.1)的序列,分别设计多组siRNA,并在北京睿博兴科生物技术有限公司合成并退火成双链。典型siRNA的序列如下表1所示。
表1:siRNA序列
表1所示的siRNA序列中,可以在RNA序列本身的3’端额外增加两个“TT”碱基作为悬垂设计,从而增加序列稳定性,在形成RICS前增加其半衰期。
本发明后续的实施例中均采用3’端增加“TT”的siRNA形式。
实施例2:siRNA转染
1.实验材料:
宿主细胞及培养条件:表达杜拉鲁肽的CHO-K1单克隆;培养基:EX-Cell Advanced CHO Fed-batch medium(sigma);实验前单克隆细胞已复苏传代一周以上,接种密度0.3-0.5×10
6个/ml,每3天传代一次,于180rpm,5%CO
2细胞培养摇床中培养;
转染培养基:Hycell TransFx-C(hyclone);
转染试剂:RNATransMate(上海生工)。
2.实验步骤:
转染前一天将单克隆细胞传代至1.0×10
6个/ml,转染当天计数,用转染培养基Hycell TransFx-C(hyclone)调整密度至2.6×10
6个/ml,分装到125ml摇瓶中,每瓶19ml。将表1中各组敲降效率最高的siRNA和转染试剂RNATrasmate分别稀释到3ml Hycell TransFx-C(hyclone)中,再将稀释后的siRNA和RNAtransmate混匀后静置5-10分钟。加入到摇瓶中的细胞悬液里,终体积为25ml,放回摇床继续培养。细胞总量、siRNA以及转染试剂用量如表2所示。
表2:细胞、siRNA以及转染试剂用量
转染后,在72h计数,将细胞悬液离心200g,10分钟,细胞用于RNA提取,RNA提取完成后逆转录为cDNA,定量检测各基因表达量以确认敲降效果;上清用于杜拉鲁肽滴度检测和纯化,纯化完成后通过HPLC肽图分析检测杜拉鲁肽蛋白羟基化修饰比例。
3.实验结果
3.1siRNA转染后72h的敲降效率:
由于PLOD1、PLOD2和PLOD3同为赖氨酸羟化酶,为查明敲降其中一个另外两个基因是否会补偿性提高表达,本实施例中单独转染其中一个PLOD的siRNA后同时对PLOD三个基因的表达水平进行检测,JMJD4则只检测自身表达。
由结果可知:单独转染si-PLOD1,PLOD1基因的表达量为对照组的32%(如图1所示);单独转染si-PLOD2,PLOD2表达量为对照组的43%(如图2所示);单独转染si-PLOD3,PLOD3表达量为对照组的40%(如图3所示);转染si-JMJD4,JMJD4表达量为对照组的41%(如图4所示),以上结果说明,siRNA达到敲降效果。并且,si-PLOD1、si-PLOD2和si-PLOD3共转染组,三个基因的表达都与对照组相比下降,分别为对照组表达量的46%、35%和42%,另外,敲降其中一个PLOD对另外两个PLOD表达量无影响(如图1、图2和图3所示)。
小结:本发明提供的siRNA分别针对4个基因PLOD1,PLOD2,PLOD3,JMJD4的敲降均能达到效果,PLOD三个基因敲降其中一个对另外两个的表达影响不大。
3.2敲降羟化酶后对细胞活细胞密度的影响
如图5所示,siRNA转染后与对照组相比,敲降PLOD1,PLOD2,PLOD3,(PLOD1+PLOD2+PLOD3)混合及JMJD4的活细胞密度略有 降低,分别为对照组的83%,91%,87%,92%和83%。
3.3敲降羟化酶后对细胞活率的影响
如图6所示,siRNA转染后与对照组相比,敲降PLOD1,PLOD2,PLOD3,(PLOD1+PLOD2+PLOD3)混合及JMJD4后,对细胞活率没有明显影响,说明敲降羟基化酶细胞也能基本维持正常生长。
3.4敲降羟化酶后对细胞单克隆产量的影响
如图7所示,siRNA转染后与对照组相比,敲降PLOD1,PLOD2,PLOD3,(PLOD1+PLOD2+PLOD3)混合及JMJD4后,蛋白产量有部分降低,分别对为对照组的69%,76%,75%,82%和84%。
3.5敲降羟化酶后对杜拉鲁肽羟基化修饰比例的影响
如图8所示,敲降PLOD2可明显降低杜拉鲁肽羟基化修饰比例,由对照组的21%降低到13%,敲降PLOD1杜拉鲁肽羟基化修饰比例为17%,敲降PLOD3和JMJD4对羟基化修饰比例没有明显影响,PLOD1、PLOD2、PLOD3共同敲降同样将羟基化修饰比例降低到13%。以上结果说明,敲降PLOD2能显著降低蛋白羟基化修饰比例。
小结:本实施例在敲降PLOD2后,活细胞密度为对照组的91%,蛋白产量为对照组的76%,但细胞活率与对照组相比没有明显差异。总体而言,采用本发明提供的方法敲降PLOD2对细胞生长、存活和蛋白产量影响不大,但可显著降低重组蛋白的羟基化修饰比例,可用于生物类似药开发中的羟基化修饰调节。
本领域技术人员可以理解,除了本发明实施例中使用的siRNA技术,在本发明中降低细胞中PLOD蛋白的表达或功能也可以通过shRNA技术或本领域已知的任何基因编辑技术来实现。示例性基因编辑技术包括规律成簇的间隔短回文重复(regular clustering of short palindrome repeats,CRISPR)、锌指核酸酶(zinc finger nuclease,ZFN)、类转录激活因子效应核酸酶(transcription activator-like effector nuclease,TALEN)技术。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
- 一种调节重组蛋白羟基化水平的方法,其特征在于,包括如下步骤:在表达重组蛋白的细胞中,降低PLOD蛋白的表达或功能;其中,所述重组蛋白不为所述PLOD蛋白。
- 根据权利要求1所述的方法,其特征在于,所述PLOD蛋白选自PLOD1、PLOD2和PLOD3中的任意一种、两种或三种;优选地,所述PLOD蛋白至少包括PLOD2。
- 根据权利要求1所述的方法,其特征在于,所述方法将干扰PLOD蛋白的表达或功能的抑制剂施用于表达重组蛋白的细胞中;所述抑制剂选自siRNA、shRNA、microRNA、反义核苷酸、核酶、编码负性突变体的核苷酸或表达载体、抗体、肽和小分子化合物;优选地,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA,优选至少包括针对PLOD2的siRNA。
- 根据权利要求3所述的方法,其特征在于,针对PLOD1的siRNA具有如SEQ ID NO.01所示的正义链和如SEQ ID NO.02所示的反义链;和/或,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08所示的反义链;和/或,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
- 根据权利要求1~4任意一项所述的方法,其特征在于,所述细胞为哺乳动物细胞,优选选自CHO细胞、HEK293细胞和Vero细胞;优选地,所述细胞为表达重组蛋白的单克隆细胞或为外源转入了重组蛋白表达载体的细胞;所述重组蛋白优选选自单克隆抗体和融合蛋白,所述融合蛋白进一步优选为Fc融合蛋白。
- 干扰PLOD蛋白的表达或功能的抑制剂,其特征在于,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA,优选至少包括针对PLOD2的siRNA;优选地,针对PLOD1的siRNA具有如SEQ ID NO.01所示的正义链和如SEQ ID NO.02所示的反义链;和/或,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08所示的反义链;和/或,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
- 一种表达重组蛋白的细胞,其特征在于,所述细胞中PLOD蛋白的表达或功能被抑制;其中,所述重组蛋白不为所述PLOD蛋白;优选地,所述PLOD蛋白选自PLOD1、PLOD2和PLOD3中的任意一种、两种或三种,优选至少包括PLOD2。
- 根据权利要求7所述的细胞,其特征在于,所述细胞中外源加入了抑制PLOD蛋白的表达或功能的抑制剂,所述抑制剂选自siRNA、shRNA、microRNA、反义核苷酸、核酶、编码负性突变体的核苷酸或表达载体、抗体、肽和小分子化合物;优选地,所述抑制剂包括针对PLOD1、PLOD2和PLOD3中的任意一种、两种或三种的siRNA,优选至少包括针对PLOD2的siRNA。
- 根据权利要求8所述的细胞,其特征在于,针对PLOD1的siRNA具有如SEQ ID NO.01所示的正义链和如SEQ ID NO.02所示的反义链;和/或,针对PLOD2的siRNA具有如SEQ ID NO.03所示的正义链和如SEQ ID NO.04所示的反义链、具有如SEQ ID NO.05所示的正义链和如SEQ ID NO.06所示的反义链或者具有如SEQ ID NO.07所示的正义链和如SEQ ID NO.08所示的反义链;和/或,针对PLOD3的siRNA具有如SEQ ID NO.09所示的正义链和如SEQ ID NO.10所示的反义链。
- 根据权利要求7~9任意一项所述的细胞,其特征在于,所述细胞为哺乳动物细胞,优选选自CHO细胞、HEK293细胞和Vero细胞;优选地,所述细胞为表达重组蛋白的单克隆细胞或为外源转入了重组蛋白表达载体的细胞;所述重组蛋白优选选自单克隆抗体和融合蛋白,所述融合蛋白进一步优选为Fc融合蛋白。
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