基于生物素誘導(dǎo)表達(dá)系統(tǒng)的HEK293細(xì)胞多基因代謝工程改造
發(fā)布時(shí)間：2019-05-15 20:29
【摘要】：目前,包括CHO、NS0、HEK293和BHK細(xì)胞等在內(nèi)的哺乳動(dòng)物細(xì)胞是生物藥物產(chǎn)業(yè)中用于生產(chǎn)需要復(fù)雜翻譯后修飾的大分子蛋白質(zhì)的首選表達(dá)系統(tǒng)。其中,來源于人胚腎的轉(zhuǎn)化細(xì)胞系HEK293細(xì)胞能夠有效水解原肽、進(jìn)行獨(dú)特的γ-羧基化修飾和完全人型糖基化,這些其他細(xì)胞無法比擬的優(yōu)點(diǎn)使其在生物藥物生產(chǎn)中日益受到重視。與其他哺乳動(dòng)物細(xì)胞表達(dá)系統(tǒng)一樣,HEK293細(xì)胞在大規(guī)模培養(yǎng)過程中的細(xì)胞凋亡和細(xì)胞過度增殖所致的目的蛋白表達(dá)效率降低的現(xiàn)象,是影響細(xì)胞表達(dá)產(chǎn)品生產(chǎn)水平和質(zhì)量的關(guān)鍵因素。 應(yīng)用代謝工程的技術(shù)手段結(jié)合基因調(diào)控表達(dá)策略,將凋亡抑制基因和細(xì)胞周期調(diào)控基因整合于哺乳動(dòng)物細(xì)胞的基因組中,定向改造哺乳動(dòng)物細(xì)胞的某些特性,賦予工程細(xì)胞在無血清培養(yǎng)過程中抵御凋亡和細(xì)胞增殖調(diào)控的體外培養(yǎng)特性,已成為哺乳動(dòng)物細(xì)胞工程領(lǐng)域中細(xì)胞系建立和優(yōu)化改造的技術(shù)發(fā)展趨勢。據(jù)此,本研究以HEK293細(xì)胞為研究對象,選取E1B-19K為凋亡抑制基因、p27~(Kip1)基因?yàn)榧?xì)胞增殖抑制基因,采用組成型過量表達(dá)E1B-19K和響應(yīng)于生物素誘導(dǎo)表達(dá)p27~(Kip1)的策略,從抗凋亡和增殖控制兩個(gè)方面對HEK293進(jìn)行基于生物素誘導(dǎo)表達(dá)的多基因代謝工程改造。 將通過重疊PCR得到的E1B-19K基因連入pcDNA3.1(+)載體,構(gòu)建E1B-19K組成型表達(dá)載體pc-E1B。以空載體轉(zhuǎn)染的細(xì)胞為對照,考察穩(wěn)定轉(zhuǎn)染pc-E1B的HEK293細(xì)胞在低葡萄糖、低血清和無谷氨酰胺三種凋亡誘導(dǎo)培養(yǎng)條件下的生長情況。E1B-19K的表達(dá)可使細(xì)胞在上述培養(yǎng)條件下的凋亡降低60-80%;同時(shí),在正常培養(yǎng)條件下,表達(dá)E1B-19K的細(xì)胞在培養(yǎng)后期的細(xì)胞活力顯著提高,衰退期延遲2 d;E1B-19K的表達(dá)對反映細(xì)胞代謝的Qglc、Qlac和Qgln等指標(biāo)無明顯影響。 從pTRE質(zhì)粒上擴(kuò)增Tet-響應(yīng)啟動(dòng)子PhCMV*-1片段,連入pc-Hy載體,構(gòu)建響應(yīng)于Dox的誘導(dǎo)表達(dá)載體pc-Hy-star;將從pCMV-SPORT6-p27上擴(kuò)增的p27~(Kip1) cDNA插入到pc-Hy-star載體的PhCMV*-1下游,構(gòu)建響應(yīng)于Dox的p27~(Kip1)誘導(dǎo)表達(dá)載體pc-Hy-star-p27。將pTet-on載體和pc-Hy-star-p27載體共轉(zhuǎn)染HEK293細(xì)胞,以細(xì)胞周期分布和活細(xì)胞密度為主要觀察指標(biāo),考察穩(wěn)定轉(zhuǎn)染的細(xì)胞在Dox誘導(dǎo)下的細(xì)胞生長;以Qglc、Qlac和Qgln為主要觀察指標(biāo),考察轉(zhuǎn)染細(xì)胞在Dox誘導(dǎo)下的細(xì)胞代謝。結(jié)果表明,p27~(Kip1)基因的表達(dá)使HEK293細(xì)胞的增殖速度顯著降低,G1期細(xì)胞比例顯著升高,葡萄糖消耗和乳酸生產(chǎn)減少。 分別自BS 168基因組克隆BS-BirA、酶切pc-Hy-GVP4質(zhì)粒獲得VP4片段,二者通過重疊PCR獲得融合轉(zhuǎn)錄激活因子BS-BirA-VP4;將BS-BirA-VP4插入到pcDNA3.1(+)載體CMV啟動(dòng)子下游,構(gòu)建調(diào)控載體pc-BS-BV;用克隆自pTRE質(zhì)粒的缺失增強(qiáng)子序列的最小CMV啟動(dòng)子PminCMV置換pc-Hy-E載體中的PCMV,得到pc-Hy-minE載體,然后將通過全合成獲得的4個(gè)串聯(lián)重復(fù)的BSOB序列插入到pc-Hy-minE載體的PminCMV上游,構(gòu)建響應(yīng)載體pc-Hy-4BSOB-minE。由此,建立了一種基于BS-BirA、響應(yīng)于生物素的新型外源基因誘導(dǎo)表達(dá)系統(tǒng)——BS-Biotin-On系統(tǒng)。在生物素存在下,BS-BirA-VP4可特異性的與BSOB結(jié)合,從而激活PminCMV下游目的基因的轉(zhuǎn)錄。將調(diào)控載體pc-BS-BV與以EGFP為目的基因的響應(yīng)載體pc-Hy-4BSOB-minE共轉(zhuǎn)染HEK293細(xì)胞,通過檢測生物素調(diào)控的EGFP的相對熒光強(qiáng)度,考察BS-Biotin-On系統(tǒng)的效果。結(jié)果表明BS-Biotin-On系統(tǒng)具有響應(yīng)迅速、背景表達(dá)較低、誘導(dǎo)效率較高等優(yōu)點(diǎn)。 將E1B-19K和BS-BirA-VP4分別插入到pBudCE4.1載體的EF-1α啟動(dòng)子和CMV啟動(dòng)子下游,構(gòu)建同時(shí)、獨(dú)立組成型表達(dá)E1B-19K和BS-BirA-VP4的載體pBud-E1B-BS-BV。將酶切自pIRES2-EGFP載體的IRES-EGFP片段,酶切自pc-Hy-star-p27載體的p27~(Kip1)片段,分別連入去除EGFP的pc-Hy-4BSOB-minE載體中,構(gòu)建響應(yīng)于生物素的p27~(Kip1)與EGFP的偶聯(lián)表達(dá)載體pc-Hy-4BSOB- minp27-IE。上述兩載體共轉(zhuǎn)染HEK293細(xì)胞,借助流式細(xì)胞儀分選和有限稀釋法進(jìn)行單克隆化,獲得了組成型表達(dá)E1B-19K和生物素調(diào)控表達(dá)p27~(Kip1)的HEK293細(xì)胞系HEK-EPIE-18。經(jīng)生物素誘導(dǎo)后,正常培養(yǎng)后期的HEK-EPIE-18細(xì)胞G1期比例由47.88%升高至66.92%;與空載體轉(zhuǎn)染的HEK293細(xì)胞(HEK-CT)相比,在廢棄培養(yǎng)基中的細(xì)胞凋亡減少47-54%。表明所構(gòu)建的HEK-EPIE-18細(xì)胞系具有抵御凋亡和細(xì)胞增殖調(diào)控的體外培養(yǎng)特性。 為驗(yàn)證多基因代謝工程改造細(xì)胞系用于外源目的蛋白表達(dá)的效果,用rhTNFR-Fc表達(dá)載體pc-TNFR-Fc分別轉(zhuǎn)染HEK-CT細(xì)胞和HEK-EPIE-18細(xì)胞,比較穩(wěn)定轉(zhuǎn)染的細(xì)胞在無血清懸浮培養(yǎng)中的Qp和rhTNFR-Fc累積濃度。在無生物素誘導(dǎo)的條件下,與轉(zhuǎn)染pc-TNFR-Fc的HEK-CT相比,轉(zhuǎn)染的HEK-EPIE-18細(xì)胞Qp無顯著差別,活細(xì)胞密度和細(xì)胞活力均顯著升高,rhTNFR-Fc累積濃度提高56.8%;在生物素誘導(dǎo)的條件下,pc-TNFR-Fc轉(zhuǎn)染的HEK-EPIE-18細(xì)胞的增殖明顯減緩、細(xì)胞活力維持在90%以上,Qp提高1倍左右,rhTNFR-Fc累積濃度比pc-TNFR-Fc轉(zhuǎn)染的HEK-CT細(xì)胞提高約1.1倍。綜合上述結(jié)果表明,選取E1B-19K為凋亡抑制基因、p27~(Kip1)為細(xì)胞增殖抑制基因,采用組成型過量表達(dá)E1B-19K和響應(yīng)于生物素誘導(dǎo)表達(dá)p27~(Kip1)的多基因代謝工程改造,是改善哺乳動(dòng)物細(xì)胞體外培養(yǎng)生物學(xué)特性、提高哺乳動(dòng)物細(xì)胞表達(dá)產(chǎn)品生產(chǎn)效率的有效途徑。
[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
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
本文編號(hào)：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
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 安芳蘭;楊保田;董文教;武發(fā)菊;宋玉霞;董金杰;牟克斌;劉學(xué)榮;;動(dòng)物細(xì)胞代謝工程進(jìn)展[J];生物技術(shù)通訊;2009年05期
2 徐莉;饒春明;;神經(jīng)生長因子的研究進(jìn)展[J];中國生物制品學(xué)雜志;2014年01期
，本文編號(hào)：2477735
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