基于生物素诱导表达系统的HEK293细胞多基因代谢工程改造
发布时间：2019-05-15 20:29
【摘要】：目前,包括CHO、NS0、HEK293和BHK细胞等在内的哺乳动物细胞是生物药物产业中用于生产需要复杂翻译后修饰的大分子蛋白质的首选表达系统。其中,来源于人胚肾的转化细胞系HEK293细胞能够有效水解原肽、进行独特的γ-羧基化修饰和完全人型糖基化,这些其他细胞无法比拟的优点使其在生物药物生产中日益受到重视。与其他哺乳动物细胞表达系统一样,HEK293细胞在大规模培养过程中的细胞凋亡和细胞过度增殖所致的目的蛋白表达效率降低的现象,是影响细胞表达产品生产水平和质量的关键因素。 应用代谢工程的技术手段结合基因调控表达策略,将凋亡抑制基因和细胞周期调控基因整合于哺乳动物细胞的基因组中,定向改造哺乳动物细胞的某些特性,赋予工程细胞在无血清培养过程中抵御凋亡和细胞增殖调控的体外培养特性,已成为哺乳动物细胞工程领域中细胞系建立和优化改造的技术发展趋势。据此,本研究以HEK293细胞为研究对象,选取E1B-19K为凋亡抑制基因、p27~(Kip1)基因为细胞增殖抑制基因,采用组成型过量表达E1B-19K和响应于生物素诱导表达p27~(Kip1)的策略,从抗凋亡和增殖控制两个方面对HEK293进行基于生物素诱导表达的多基因代谢工程改造。 将通过重叠PCR得到的E1B-19K基因连入pcDNA3.1(+)载体,构建E1B-19K组成型表达载体pc-E1B。以空载体转染的细胞为对照,考察稳定转染pc-E1B的HEK293细胞在低葡萄糖、低血清和无谷氨酰胺三种凋亡诱导培养条件下的生长情况。E1B-19K的表达可使细胞在上述培养条件下的凋亡降低60-80%;同时,在正常培养条件下,表达E1B-19K的细胞在培养后期的细胞活力显著提高,衰退期延迟2 d;E1B-19K的表达对反映细胞代谢的Qglc、Qlac和Qgln等指标无明显影响。 从pTRE质粒上扩增Tet-响应启动子PhCMV*-1片段,连入pc-Hy载体,构建响应于Dox的诱导表达载体pc-Hy-star;将从pCMV-SPORT6-p27上扩增的p27~(Kip1) cDNA插入到pc-Hy-star载体的PhCMV*-1下游,构建响应于Dox的p27~(Kip1)诱导表达载体pc-Hy-star-p27。将pTet-on载体和pc-Hy-star-p27载体共转染HEK293细胞,以细胞周期分布和活细胞密度为主要观察指标,考察稳定转染的细胞在Dox诱导下的细胞生长;以Qglc、Qlac和Qgln为主要观察指标,考察转染细胞在Dox诱导下的细胞代谢。结果表明,p27~(Kip1)基因的表达使HEK293细胞的增殖速度显著降低,G1期细胞比例显著升高,葡萄糖消耗和乳酸生产减少。 分别自BS 168基因组克隆BS-BirA、酶切pc-Hy-GVP4质粒获得VP4片段,二者通过重叠PCR获得融合转录激活因子BS-BirA-VP4;将BS-BirA-VP4插入到pcDNA3.1(+)载体CMV启动子下游,构建调控载体pc-BS-BV;用克隆自pTRE质粒的缺失增强子序列的最小CMV启动子PminCMV置换pc-Hy-E载体中的PCMV,得到pc-Hy-minE载体,然后将通过全合成获得的4个串联重复的BSOB序列插入到pc-Hy-minE载体的PminCMV上游,构建响应载体pc-Hy-4BSOB-minE。由此,建立了一种基于BS-BirA、响应于生物素的新型外源基因诱导表达系统——BS-Biotin-On系统。在生物素存在下,BS-BirA-VP4可特异性的与BSOB结合,从而激活PminCMV下游目的基因的转录。将调控载体pc-BS-BV与以EGFP为目的基因的响应载体pc-Hy-4BSOB-minE共转染HEK293细胞,通过检测生物素调控的EGFP的相对荧光强度,考察BS-Biotin-On系统的效果。结果表明BS-Biotin-On系统具有响应迅速、背景表达较低、诱导效率较高等优点。 将E1B-19K和BS-BirA-VP4分别插入到pBudCE4.1载体的EF-1α启动子和CMV启动子下游,构建同时、独立组成型表达E1B-19K和BS-BirA-VP4的载体pBud-E1B-BS-BV。将酶切自pIRES2-EGFP载体的IRES-EGFP片段,酶切自pc-Hy-star-p27载体的p27~(Kip1)片段,分别连入去除EGFP的pc-Hy-4BSOB-minE载体中,构建响应于生物素的p27~(Kip1)与EGFP的偶联表达载体pc-Hy-4BSOB- minp27-IE。上述两载体共转染HEK293细胞,借助流式细胞仪分选和有限稀释法进行单克隆化,获得了组成型表达E1B-19K和生物素调控表达p27~(Kip1)的HEK293细胞系HEK-EPIE-18。经生物素诱导后,正常培养后期的HEK-EPIE-18细胞G1期比例由47.88%升高至66.92%;与空载体转染的HEK293细胞(HEK-CT)相比,在废弃培养基中的细胞凋亡减少47-54%。表明所构建的HEK-EPIE-18细胞系具有抵御凋亡和细胞增殖调控的体外培养特性。 为验证多基因代谢工程改造细胞系用于外源目的蛋白表达的效果,用rhTNFR-Fc表达载体pc-TNFR-Fc分别转染HEK-CT细胞和HEK-EPIE-18细胞,比较稳定转染的细胞在无血清悬浮培养中的Qp和rhTNFR-Fc累积浓度。在无生物素诱导的条件下,与转染pc-TNFR-Fc的HEK-CT相比,转染的HEK-EPIE-18细胞Qp无显著差别,活细胞密度和细胞活力均显著升高,rhTNFR-Fc累积浓度提高56.8%;在生物素诱导的条件下,pc-TNFR-Fc转染的HEK-EPIE-18细胞的增殖明显减缓、细胞活力维持在90%以上,Qp提高1倍左右,rhTNFR-Fc累积浓度比pc-TNFR-Fc转染的HEK-CT细胞提高约1.1倍。综合上述结果表明,选取E1B-19K为凋亡抑制基因、p27~(Kip1)为细胞增殖抑制基因,采用组成型过量表达E1B-19K和响应于生物素诱导表达p27~(Kip1)的多基因代谢工程改造,是改善哺乳动物细胞体外培养生物学特性、提高哺乳动物细胞表达产品生产效率的有效途径。
[Abstract]:Currently, mammalian cells, including CHO, NS0, HEK293, and BHK cells, are preferred expression systems for producing macromolecular proteins that require complex translation after complex translation. Among them, the human embryonic kidney-derived transformed cell line HEK293 cell is capable of effectively hydrolyzing the propeptide, carrying out unique modification and complete human-type glycosylation, and the other cells can not be compared with the other cells, so that the human embryonic kidney-derived transformed cell line HEK293 cell is more and more important in the production of biological drugs. Like other mammalian cell expression systems, the decrease in the efficiency of the expression of the target protein, which is caused by the apoptosis of the HEK293 cells in the large-scale culture, and the excessive proliferation of the cells, is a key factor that affects the production level and quality of the cell-expressing products. By using the technical means of the metabolic engineering to combine the gene regulation and expression strategy, the apoptosis-inhibiting gene and the cell cycle regulation gene are integrated in the genome of the mammalian cell, and the directional modification of some of the mammalian cells The in vitro culture characteristics of cell line establishment and optimization transformation in the field of mammalian cell engineering have become the trend of the development of cell line in the field of mammalian cell engineering. In this study, E1B-19K was selected as the apoptosis-inhibiting gene, and p27 ~ (Kip1) gene was the cell-proliferation-inhibiting gene. E1B-19K and p27 ~ (Kip1) were expressed in response to biotin-induced expression of E1B-19K and in response to biotin-induced expression of p27 ~ (Kip1). Modification of HEK293 Based on Biotin-induced Expression of HEK293 in Two Aspects of Anti-apoptotic and Proliferative Control and the E1B-19K gene obtained by the overlapping PCR is connected into the pcDNA3.1 (+) vector to construct an E1B-19K constitutive expression vector pc- E1B. The cells transfected with empty vector were control, and the growth of HEK293 cells stably transfected with pc-E1B was investigated under the conditions of low glucose, low serum and no-glutamate-free amine. The expression of E1B-19K could decrease the cell viability in the late stage of culture. There are no clear indicators such as glc, Qllac and Qgln. The p27 ~ (Kip1) cDNA amplified from pCMV-SPORT6-p27 was inserted into the downstream of PhCMV *-1 of the pc-Hy-star vector to construct p27 ~ (Kip1)-induced expression vector pc-Hy-sta in response to Dox. R-p27. pTet-on vector and pc-Hy-star-p27 vector were co-transfected into HEK293 cells to observe the cell cycle distribution and the cell density as the main observation index. The results showed that the expression of p27 ~ (Kip1) gene significantly decreased the proliferation rate of HEK293 cells, and the proportion of cells in G1 phase increased significantly. The acid production was reduced. The VP4 fragment was obtained from the BS-BirA, the enzyme-cut pc-Hy-GVP4 plasmid from the genome of the BS 168, the fusion transcription activation factor BS-BirA-VP4 was obtained by overlapping PCR, and the BS-BirA-VP4 was inserted downstream of the pcDNA3.1 (+) vector CMV promoter to construct the regulatory vector p. c-BS-BV; replacing the PCMV in the pc-Hy-E vector with the minimum CMV promoter PminCMV of the deletion enhancer sequence cloned from the pTRE plasmid to obtain a pc-Hy-minE vector, and then inserting the four serially-repeated BSOB sequences obtained by full-synthesis into the PminCMV upstream of the pc-Hy-minE vector to construct the response vector pc-Hy-4BS In this way, a new exogenous gene-induced expression system, BS-Biot, based on BS-BirA, in response to biotin, was established. in-On system. BS-BirA-VP4 binds specifically to BSOB in the presence of biotin, thereby activating the downstream of PminCMV A control vector pc-BS-BV was co-transfected with a response vector pc-Hy-4BSOB-minE with EGFP as a target gene, and the HEK293 cell was co-transfected with a response vector pc-Hy-4BSOB-minE with EGFP as a target gene, and the BS-Biotin-BV was examined by detecting the relative fluorescence intensity of the biotin-regulated EGFP. The results show that the BS-Biotin-On system has a rapid response and low background expression. The E1B-19K and the BS-BirA-VP4 are respectively inserted into the EF-1 promoter of the pBudCE4.1 vector and the downstream of the CMV promoter, and the vector pBud-19K and the carrier pBud of the BS-BirA-VP4 are constructed independently. E1B-BS-BV. The p27 ~ (Kip1) fragment of pIRES2-EGFP vector was cut from the pIRES2-EGFP vector, and the p27 ~ (Kip1) fragment of the p27 ~ (Kip1) and EGFP was respectively connected to the p27 ~ (Kip1) and EGFP of the p27 ~ (Kip1) and EGFP. The two vectors were co-transfected with HEK293 cells, and by means of flow cytometry and limited dilution, the HEK293 cell line HE was obtained by the expression of E1B-19K and biotin-regulated expression p27 ~ (Kip1). K-EPIE-18. The G1 phase of HEK-EPIE-18 cells was increased from 47.88% to 66.92% in the late stage of normal culture, compared with HEK293 cells transfected with empty vector (HEK-CT). The death was reduced by 47-54%. It was shown that the constructed HEK-EPIE-18 cell line was resistant to apoptosis and cell proliferation. In order to verify the effect of the multi-gene metabolic engineering transformation cell line for the expression of foreign object protein, the expression vector pc-TNFR-Fc was transfected into HEK-CT cells and HEK-EPIE-18 cells by using the rhTNFR-Fc expression vector pc-TNFR-Fc, and the stably transfected cells were Qp and rh in the serum-free suspension culture. The cumulative concentration of TNFR-Fc. Compared with the HEK-CT transfected with the pc-TNFR-Fc, the transfected HEK-EPIE-18 cells Qp had no significant difference compared with the HEK-CT transfected with the pc-TNFR-Fc. The cumulative concentration of the rhTNFR-Fc increased by 56.8% compared with the HEK-CT transfected with the pc-TNFR-Fc, and the HEK-EPIE-Fc transfected with the pc-TNFR-Fc under the condition of biotin induction- The proliferation of 18 cells was significantly reduced, the cell viability was maintained above 90%, the Qp was increased by about 1-fold, and the cumulative concentration of rhTNFR-Fc was higher than that of the pc-TNFR-Fc-transfected HEK- The results show that E1B-19K is the apoptosis-inhibiting gene, p27 ~ (Kip1) is the cell proliferation-inhibiting gene, and the constitutive overexpression of E1B-19K and the transformation of the multi-gene metabolic engineering in response to the expression of p27 ~ (Kip1) in response to the biotin-induced expression of p27 ~ (Kip1) are the improvement of the mammals. Cell Culture in Vitro to Improve the Expression of Mammalian Cells
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
本文编号：2477735
[Abstract]:Currently, mammalian cells, including CHO, NS0, HEK293, and BHK cells, are preferred expression systems for producing macromolecular proteins that require complex translation after complex translation. Among them, the human embryonic kidney-derived transformed cell line HEK293 cell is capable of effectively hydrolyzing the propeptide, carrying out unique modification and complete human-type glycosylation, and the other cells can not be compared with the other cells, so that the human embryonic kidney-derived transformed cell line HEK293 cell is more and more important in the production of biological drugs. Like other mammalian cell expression systems, the decrease in the efficiency of the expression of the target protein, which is caused by the apoptosis of the HEK293 cells in the large-scale culture, and the excessive proliferation of the cells, is a key factor that affects the production level and quality of the cell-expressing products. By using the technical means of the metabolic engineering to combine the gene regulation and expression strategy, the apoptosis-inhibiting gene and the cell cycle regulation gene are integrated in the genome of the mammalian cell, and the directional modification of some of the mammalian cells The in vitro culture characteristics of cell line establishment and optimization transformation in the field of mammalian cell engineering have become the trend of the development of cell line in the field of mammalian cell engineering. In this study, E1B-19K was selected as the apoptosis-inhibiting gene, and p27 ~ (Kip1) gene was the cell-proliferation-inhibiting gene. E1B-19K and p27 ~ (Kip1) were expressed in response to biotin-induced expression of E1B-19K and in response to biotin-induced expression of p27 ~ (Kip1). Modification of HEK293 Based on Biotin-induced Expression of HEK293 in Two Aspects of Anti-apoptotic and Proliferative Control and the E1B-19K gene obtained by the overlapping PCR is connected into the pcDNA3.1 (+) vector to construct an E1B-19K constitutive expression vector pc- E1B. The cells transfected with empty vector were control, and the growth of HEK293 cells stably transfected with pc-E1B was investigated under the conditions of low glucose, low serum and no-glutamate-free amine. The expression of E1B-19K could decrease the cell viability in the late stage of culture. There are no clear indicators such as glc, Qllac and Qgln. The p27 ~ (Kip1) cDNA amplified from pCMV-SPORT6-p27 was inserted into the downstream of PhCMV *-1 of the pc-Hy-star vector to construct p27 ~ (Kip1)-induced expression vector pc-Hy-sta in response to Dox. R-p27. pTet-on vector and pc-Hy-star-p27 vector were co-transfected into HEK293 cells to observe the cell cycle distribution and the cell density as the main observation index. The results showed that the expression of p27 ~ (Kip1) gene significantly decreased the proliferation rate of HEK293 cells, and the proportion of cells in G1 phase increased significantly. The acid production was reduced. The VP4 fragment was obtained from the BS-BirA, the enzyme-cut pc-Hy-GVP4 plasmid from the genome of the BS 168, the fusion transcription activation factor BS-BirA-VP4 was obtained by overlapping PCR, and the BS-BirA-VP4 was inserted downstream of the pcDNA3.1 (+) vector CMV promoter to construct the regulatory vector p. c-BS-BV; replacing the PCMV in the pc-Hy-E vector with the minimum CMV promoter PminCMV of the deletion enhancer sequence cloned from the pTRE plasmid to obtain a pc-Hy-minE vector, and then inserting the four serially-repeated BSOB sequences obtained by full-synthesis into the PminCMV upstream of the pc-Hy-minE vector to construct the response vector pc-Hy-4BS In this way, a new exogenous gene-induced expression system, BS-Biot, based on BS-BirA, in response to biotin, was established. in-On system. BS-BirA-VP4 binds specifically to BSOB in the presence of biotin, thereby activating the downstream of PminCMV A control vector pc-BS-BV was co-transfected with a response vector pc-Hy-4BSOB-minE with EGFP as a target gene, and the HEK293 cell was co-transfected with a response vector pc-Hy-4BSOB-minE with EGFP as a target gene, and the BS-Biotin-BV was examined by detecting the relative fluorescence intensity of the biotin-regulated EGFP. The results show that the BS-Biotin-On system has a rapid response and low background expression. The E1B-19K and the BS-BirA-VP4 are respectively inserted into the EF-1 promoter of the pBudCE4.1 vector and the downstream of the CMV promoter, and the vector pBud-19K and the carrier pBud of the BS-BirA-VP4 are constructed independently. E1B-BS-BV. The p27 ~ (Kip1) fragment of pIRES2-EGFP vector was cut from the pIRES2-EGFP vector, and the p27 ~ (Kip1) fragment of the p27 ~ (Kip1) and EGFP was respectively connected to the p27 ~ (Kip1) and EGFP of the p27 ~ (Kip1) and EGFP. The two vectors were co-transfected with HEK293 cells, and by means of flow cytometry and limited dilution, the HEK293 cell line HE was obtained by the expression of E1B-19K and biotin-regulated expression p27 ~ (Kip1). K-EPIE-18. The G1 phase of HEK-EPIE-18 cells was increased from 47.88% to 66.92% in the late stage of normal culture, compared with HEK293 cells transfected with empty vector (HEK-CT). The death was reduced by 47-54%. It was shown that the constructed HEK-EPIE-18 cell line was resistant to apoptosis and cell proliferation. In order to verify the effect of the multi-gene metabolic engineering transformation cell line for the expression of foreign object protein, the expression vector pc-TNFR-Fc was transfected into HEK-CT cells and HEK-EPIE-18 cells by using the rhTNFR-Fc expression vector pc-TNFR-Fc, and the stably transfected cells were Qp and rh in the serum-free suspension culture. The cumulative concentration of TNFR-Fc. Compared with the HEK-CT transfected with the pc-TNFR-Fc, the transfected HEK-EPIE-18 cells Qp had no significant difference compared with the HEK-CT transfected with the pc-TNFR-Fc. The cumulative concentration of the rhTNFR-Fc increased by 56.8% compared with the HEK-CT transfected with the pc-TNFR-Fc, and the HEK-EPIE-Fc transfected with the pc-TNFR-Fc under the condition of biotin induction- The proliferation of 18 cells was significantly reduced, the cell viability was maintained above 90%, the Qp was increased by about 1-fold, and the cumulative concentration of rhTNFR-Fc was higher than that of the pc-TNFR-Fc-transfected HEK- The results show that E1B-19K is the apoptosis-inhibiting gene, p27 ~ (Kip1) is the cell proliferation-inhibiting gene, and the constitutive overexpression of E1B-19K and the transformation of the multi-gene metabolic engineering in response to the expression of p27 ~ (Kip1) in response to the biotin-induced expression of p27 ~ (Kip1) are the improvement of the mammals. Cell Culture in Vitro to Improve the Expression of Mammalian Cells
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
【共引文献】
相关期刊论文 前2条
1 安芳兰;杨保田;董文教;武发菊;宋玉霞;董金杰;牟克斌;刘学荣;;动物细胞代谢工程进展[J];生物技术通讯;2009年05期
2 徐莉;饶春明;;神经生长因子的研究进展[J];中国生物制品学杂志;2014年01期
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