重组长效EPO（NESP）高效表达细胞株的构建
摘要
本文首先将pFRT/lacZeo质粒转染CHO-K1细胞,经半乳糖苷酶活性检测筛选得到了含高活性位点的细胞株CHO/FRT2.通过定点突变对NESPl(专利报道序列)序列进行了密码子优化,得到了NESP2、NESP3,并构建了相关的表达载体pcDNA5/FRT/NESP。将表达载体与含FLP酶基因的pOG44质粒共转CHO/FRT2细胞,得到了高效表达重组NESP的细胞株CHO/FRT2/NESP:NESP1、NESP2、NESP3的表达水平分别为786 IU/ml、1315 IU/ml、1029 IU/ml,表明经过密码子优化的NESP2具有更高的蛋白表达水平。
此外,将载体pcDNA5/FRT/NESP2转染各CHO/FRT细胞,ELISA检测结果显示NESP2的表达与半乳糖苷酶活性水平趋势相同,证实半乳糖苷酶作为筛选指示蛋白,可以用来筛选有效的蛋白表达热区。
之后,通过蓝胶亲和层析,凝胶过滤,离子交换层析纯化了重组CHO/FRT2/NESP2细胞培养上清,得到了一定量初步纯化的重组NESP蛋白样品。通过SDS-PAGE电泳和Western blot分析证实,相对于原型EPO,NESP具有更高的分子量。等电聚焦分析结果也进一步证实NESP比EPO具有更高的唾液酸丰度,糖基化程度更高。
The pFRT/lacZeo plasmid was transfected into CHO-K1 cells, and then obtained the CHO/FRT2 with high-level active domain by detecting the activity ofβ-galactosidase. NESP2 and NESP3 were obtained by codon optimization of NESP1 (the patent reported sequence) using site-directed mutation method, and then expression vector pcDNA5/FRT/NESP were constructed. The CHO/FRT2/NESP expressing high-level NESP was obtained by cotransfecting the expression vector pOG44 with flp gene into the CHO/FRT2. The expression-level of NESP1, NESP2 and NESP3 were 786 IU/ml,1315 IU/ml and 1029 IU/ml, respectively. The results indicated that codon optimization could improve the NESP expression.
The expression vector pcDNA5/FRT/NESP2 was transfected into CHO/FRT cell lines, the results of ELISA demonstrated that expression of NESP2 and activity ofβ-galactosidase maintained the same trend, which indicated thatβ-galactosidase can be used as a good reporter to screen hot-spots of high-level protein expression.
Then the cultured supernatant of CHO/FRT2/NESP2 was purified by blue-gel affinity chromatography, gel filtration chromatography (GFC) and ion exchange chromatography (IEC), SDS-PAGE and Western blot analysis indicated that compared with rHuEPO, NESP had a higher molecular weight. The result of isoelectrofocusing demonstrated that the most basic isoform of NESP with increased negative charge was more acidic than the most acidic isoform of rHuEPO.
此外,将载体pcDNA5/FRT/NESP2转染各CHO/FRT细胞,ELISA检测结果显示NESP2的表达与半乳糖苷酶活性水平趋势相同,证实半乳糖苷酶作为筛选指示蛋白,可以用来筛选有效的蛋白表达热区。
之后,通过蓝胶亲和层析,凝胶过滤,离子交换层析纯化了重组CHO/FRT2/NESP2细胞培养上清,得到了一定量初步纯化的重组NESP蛋白样品。通过SDS-PAGE电泳和Western blot分析证实,相对于原型EPO,NESP具有更高的分子量。等电聚焦分析结果也进一步证实NESP比EPO具有更高的唾液酸丰度,糖基化程度更高。
The pFRT/lacZeo plasmid was transfected into CHO-K1 cells, and then obtained the CHO/FRT2 with high-level active domain by detecting the activity ofβ-galactosidase. NESP2 and NESP3 were obtained by codon optimization of NESP1 (the patent reported sequence) using site-directed mutation method, and then expression vector pcDNA5/FRT/NESP were constructed. The CHO/FRT2/NESP expressing high-level NESP was obtained by cotransfecting the expression vector pOG44 with flp gene into the CHO/FRT2. The expression-level of NESP1, NESP2 and NESP3 were 786 IU/ml,1315 IU/ml and 1029 IU/ml, respectively. The results indicated that codon optimization could improve the NESP expression.
The expression vector pcDNA5/FRT/NESP2 was transfected into CHO/FRT cell lines, the results of ELISA demonstrated that expression of NESP2 and activity ofβ-galactosidase maintained the same trend, which indicated thatβ-galactosidase can be used as a good reporter to screen hot-spots of high-level protein expression.
Then the cultured supernatant of CHO/FRT2/NESP2 was purified by blue-gel affinity chromatography, gel filtration chromatography (GFC) and ion exchange chromatography (IEC), SDS-PAGE and Western blot analysis indicated that compared with rHuEPO, NESP had a higher molecular weight. The result of isoelectrofocusing demonstrated that the most basic isoform of NESP with increased negative charge was more acidic than the most acidic isoform of rHuEPO.
引文
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[2]Jacobs K, Shoemaker C, Rudersdorf R, Neill SD, Kaufman RJ, Mufson A, Seehra A, Jones SS, Hewick R, Fritsch EF, Kawakita M, Shimiza T, Miyoke T. Isolation and characterization of genomic cDNA clones of human erythropoietin. Nature.1985, 313:806-810
[3]Lin FK, Suggs S, Lin CH, Browne JK, Smailing R, Egric JC, Chen KK, Fox GM, Martin F, Wasser Z. Cloning and expression of the human erythropoietin gene. Proc. Natl. Acad. Sci.1985,92:7850-7884
[4]晋晓春.红细胞生成素.生理科学进展.1995,26(1)：73-77
[5]Long D.L, Doherty D.H, Eisenberg S.P, Smith D.J, Rosendahl M.S, Christensen K.R, Edwards D.P, Chlippala E.A, Cox G.N. Design of homogeneousmonopegylated erythropoietin analogs with preserved in vitro bioactivity, Exp.Hematol.2006, 34:697-794.
[6]Takeachi M, Takasaki S, Shimada M, Kobata A. Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J. Boil. Chem.1990,265:12127-12130
[7]Koury MJ, Bondurant MC. Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells. J Cell Physiol.1988,137:65-74
[8]MJ Koury, MC Bondurant. Erythropoietin retards DNA breakdown and prevents programmed death in crythroid progenitor cells. Science.1990,248:378-381
[9]李宓,邹和群.促红细胞生成素对CRF竭患者免疫功能的影响.中国血液净化.2005,4(4)：193-196
[10]Ribatti D, Presta M, Vacca A, Ria R, Giuliani R, Dell'Era P, Nico B, Roncali L, Dammacco F. Human erythropoietin induces a pro-anrgiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo. Blood.1999,93:26-27
[11]Sakanaka M, Wen TC, Matsuda S, Masuda S. Morishita E, Nagao M,Sasaki R. In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc. Natl. Acad. Sci.1998,95 (8):4635-4640
[12]Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichehi C. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord lxauma. Proc. Natl. Acad. Sci.2002, 99:9450-9455
[13]Thibault H, Galan P, Selz F, Preziosi P, Olivier C. The immune response in iron-deficient young children. effect of iron supplementation on cell-mediated immunity. Eur. J. Pediatr.1993,152 (1):120-124
[14]Feizi T, Mulloy B. Carbohydrates and glycoconjugates:Glycomics:the new era of carbohydrate biology. Curr. Opin. Struct. Biol.2003,13(5):602-604
[15][美]A.瓦尔基等编著,张树政,朱正美,王克夷等译校.糖生物学基础.北京：科学出版社,2003
[16]Blithe DL. Biological functions of oligosaccharides on glycoproteins. Trends G lycosci. Glycotechnol.1993,5:81-98
[17]Dwek RA. Glycobiology:towards understanding the function of the sugars. Biochem. Soc. Trans.1995,23:1-25
[18]Alison J et al. Biotechnol Bioeng,2000; 67:544
[19]Sambrook J et al. Molecular Cloning. (2nd ed) Science press,1998.765786
[20]Rolf GW et al. Drug Res,1998;48 (Ⅱ):870
[21]Kanfman RJ.Methods Mol Biol,1997;62:287
[22]Studier FW et al.J Mol Biol,1986;189:113
[23]Takeuchi M, Kobata A. Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology.1991,1:337-346
[24]Grosveld, F. et al. Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell.1987,51:975-985
[25]Louise M. Barnes, Catherine M. Bentley, Alan J. Dickson. Advances in animal cell recombinant protein production:GS-NS0 expression system. Cytotechnology.2002,2, 109-123
[26]Mielke, C. et al. Anatomy of highly expressing chromosomal sites targeted by retroviral vectors. Biochemistry.1996,35:2239-2252
[27]Koduri, R.K. et al. An efficient homologous recombination vector pTV(Ⅰ) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells. Gene.2001,280:87-95
[28]Li, Q. et al. Locus control regions. Blood.2002,100:3077-3086
[29]Sabbattini, P. et al. Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the lambda5-VpreBl locus control region. Mol.Cell. Biol.1999,19,:671-679
[30]Shewchuk, B.M. et al. Specification of unique Pit-1 activity in the hGH locus control region. Proc. Natl. Acad. Sci. U. S. A.1999,99:11784-11789
[31]Chung, J.H. et al. A element of the chicken beta-globindomain serves as an insulator in human erythroid cells and protects against position effects in Drosophila. Cell 1993, 74:505-514
[32]Izumi, M, Gilbert, D.M. Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts. J. Cell.Biochem.1999,76:280-289
[33]Williams, S. et al. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mamma-lian cells. BMC Biotechnol.2005,5:17
[34]Benton, T. et al. The use of UCOE vectors in combination with a preadapted serum-free suspension cell line allows for rapid production of large quantities of protein. Cytotechnology 2002,38:43-46
[35]Kim, J.M. et al. Improved recombinant gene expression in CHO cells using matrix attachment regions. J. Biotechnol.2004,107:95-105
[36]Alt F W, Kellems R E, Bertino J R. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem 1978, 253:1357-1370
[37]Urlaub G, Chasin L A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci 1980,77:4216-4220
[38]Hammill L, Welles J, Carson G R. The gel microdrop secretion assay:Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells. Cytotechnology 2000,34:27-37
[39]Barnes L M, Bentley C M, Dickson A J. Advances in animal cell recombinant protein production:GS-NS0 expression system.Cytotechnology.2000,32:109-123
[40]Feng B, Shiber S K, Max S R.Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture. J Cell Physiol.1990,145:376-380
[41]Eisenberg D, Gill H S, Pfluegl G M U, Rotstein S H. Structure-Function Relationships in glutamine synthetase. Biochim Biophys Acta 2000,1477:122-145
[42]Brown M E, Renner G, Field R P, Hassell T. Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system. Cytotechnology 1992,9:231-236
[43]Bebbington C R, Renner G, Thomson S, King D, Abrams D, Yarranton G T. High-Level Expression of a Recombinant Antibody from myeloma cells using a Glutamine Synthetase Gene as an Amplifiable Selectable Marker. Bio/Technol 1992, 10:169-175
[44]Kadesch T, Berg P. Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid. Mol Cell Biol.1986, 6(7):2593-2601
[45]Proudfoot N J. Transcriptional interference and termination between duplicated α-globin gene constructs suggests a novel mechanism for gene regulation. Nature 1986,322,562-565
[46]Simonsen C C, McGrogan M. The molecular biology of production cell lines. Biologicals 1994,22:85-94
[47]Keen M J, Hale C. The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker. Cytotechnology 1996,18:207-217
[48]Hodgson J. Expression systems:a user's guide. Bio/Technol 1993,11:887-893
[49]Bebbington C R., Renner G, Thomson S, King D, Abrams D, Yarranton G T. High-Level expression of a recombinant antibody from, myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Nature Biotechnology.1992,10:169-175
[50]Hsieh P, Resher MR, Robbins PW. Host-dependent variation of asparagine linked oligosaccharides at individual glycosylation sites of Sindbis virus glycoproteins. J. Biol. Chem.1983,258:2548-2554
[51]Hammill L, Welles J, Carson, GR. The gel microdrop secretion assay:Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells. Cytotechnology 2000,34:27-37
[52]Brich J R, Bebbington C R, Field R, Renner G, Brand H, Finney H, The production of recombinant antibodies using the glutamine synthetase(GS)system. In:Kaminogawa S, Ametani A, Hachimura S. Animal Technology:Basic and Applied Aspects, Kluwer Academic Publishers, Doudrecht.1993,Vol.5, p573-577
[53]Broach J R., Guarascio V R, Jayaram M. Recombination within the yeast plasmid 2μ circle is site-specific. Cell,1982,29:227-234.
[54]Cox M M. The FLP protein of the yeast 2 μm plasmid:expression of a eukaryotic genetic recombination system in Escherichia coli. Proc. Natl. Acad. Sci. USA,1983,80: 4223-4227.
[55]Vetter D, Andrews B J, Roberts-Beatty L, Sadowski P D. Site-specific recombination of yeast 2-μm DNA in vitro. Proc. Natl. Acad. Sci. USA,1983,80:7284-7288.
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