细胞饼瞬时表达体系的优化
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摘要
细胞饼瞬时转化技术具有重要的应用前景,然而其转化效率的优化研究尚未见报道。通过农杆菌介导法,系统地比较不同农杆菌浓度,抗氧化剂以及表面活性剂的添加对细胞饼瞬时转化效率的影响。研究结果表明:1)侵染用农杆菌的浓度在较高水平(OD=1.0)时更有利于转化效率的提升;2)两种抗氧化剂中,辛硫酸效果较半胱氨酸为好。但两者均表现为低浓度对转化效率具有促进作用,而高浓度则表现为抑制作用;3)测试的两种表面活性剂,Triton X-100以及Silwet L-77对转化效率均具有显著提升作用,而Silwet L-77的作用则更为突出;4)在优化条件下,人白介素10的含量较对照高出3.66倍。本研究的结果可进一步提升细胞饼瞬时转化技术的应用前景,为后续这一技术的应用奠定了一定的理论基础。
The transient expression of cell pack has important application prospects, but the optimization of its transformation efficiency has not been reported yet. In this study, the effects of different concentrations of Agrobacterium tumefaciens, antioxidant and surfactant on transient expression efficiency of cell pack were systematically compared by A. tumefaciens-mediated transformation. The results showed that: 1. The concentration of A.tumefaciens for infection was more conducive to the improvement of transformation efficiency at a high level(OD=1.0); 2. Lipoic acid showed better performance than that of cysteine. Low concentration of antioxidant promoted the transformation efficiency, while high concentration showed inhibition effects; 3. Both of the tested surfactants,Triton X-100 and Silwet L-77, had significant improvement on the transformation efficiency. While, the effect of Silwet L-77 was more prominent. 4. Under the optimized conditions, the content of human interleukin-10 was3.66 times higher than that of the control. The results of this study can further enhance the application prospects of transient cell cake transformation technology, and lay a theoretical foundation for the follow-up application of this technology.
The transient expression of cell pack has important application prospects, but the optimization of its transformation efficiency has not been reported yet. In this study, the effects of different concentrations of Agrobacterium tumefaciens, antioxidant and surfactant on transient expression efficiency of cell pack were systematically compared by A. tumefaciens-mediated transformation. The results showed that: 1. The concentration of A.tumefaciens for infection was more conducive to the improvement of transformation efficiency at a high level(OD=1.0); 2. Lipoic acid showed better performance than that of cysteine. Low concentration of antioxidant promoted the transformation efficiency, while high concentration showed inhibition effects; 3. Both of the tested surfactants,Triton X-100 and Silwet L-77, had significant improvement on the transformation efficiency. While, the effect of Silwet L-77 was more prominent. 4. Under the optimized conditions, the content of human interleukin-10 was3.66 times higher than that of the control. The results of this study can further enhance the application prospects of transient cell cake transformation technology, and lay a theoretical foundation for the follow-up application of this technology.
引文
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[16]DAN Y.Biological functions of antioxidants in plant transformation[J].In Vitro Cellular&Developmental BiologyPlant,2008,44(3):149-161.
[17]LI S,CONG Y,LIU Y,et al.Optimization of Agrobacterium-Mediated Transformation in Soybean[J].Frontiers in Plant Science,2017(24):246-254.
[18]WROBLEWSKI T,TOMCZAK A,MICHELMORE R.Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce,tomato and Arabidopsis[J].Plant Biotechnology Journal,2005,3(2):259-273.
[19]KOMAROVA T V,BASCHIERI S,DONINI M,et al.Transient expression systems for plant-derived biopharmaceuticals[J].Expert Review of Vaccines,2010,9(8):859-876.
[20]HAHN S,GIRITCH A,BARTELS D,et al.A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants[J].Plant Biotechnology Journal,2015,13(5):708-716.
[2]张晓筱.单克隆抗体研究进展[J].贵州教育学院学报(自然科学),2007,18(4):56-61.
[3]CHEN Q.Recombinant Therapeutic Molecules Produced in Plants[M].Jacqueline MacDonald:Advances in Botanical Research.Academic Press,2018:207-244.
[4]HELLWIG S,DROSSARD J,TWYMAN R M,et al.Plant cell cultures for the production of recombinant proteins[J].Nature biotechnology,2004,22(11):1415-1422.
[5]CHEN Q,LAI H.Gene delivery into plant cells for recombinant protein production[J].BioMed research international,2014(11):342-351.
[6]NAJI-TALAKAR S.Plant derived biopharmaceuticals:overview and success of agroinfiltration[J].Trends Capstone,2017,2(1):12.
[7]BAYER E,THOMAS C L,MAULE A J.Plasmodesmata in Arabidopsis thaliana suspension cells[J].Protoplasma,2004,223(2-4):93.
[8]SUKENIK S,KARUPPANAN K,LI Q,et al.Transient Recombinant Protein Production in GlycoengineeredNicotiana benthamiana Cell Suspension Culture[J].International Journal of Molecular Sciences,2018,19(4):1205.
[9]RADEMACHER T.Method for the generation and cultivation of a plant cell pack:USA,20140342455[P].2014.
[10]SHAMLOUL M,TRUSA J,METT V,et al.Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana[J].Journal of visualized experiments,2014(86):51204.
[11]JOH L D,WROBLEWSKI T,EWING N N,et al.High-level transient expression of recombinant protein in lettuce[J].Biotechnology and Bioengineering,2005,91(7):861-871.
[12]KIM M J,BAEK K,PARK C-M.Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis[J].Plant Cell Reports,2009,28(8):1159-1167.
[13]LI J,CHEN M,LIU X,et al.A Highly Efficient System Establishment of Transient Expression in Lettuce[J].Acta Horticulturae Sinica,2006,33(2):405-407.
[14]BORTESI L,ROSSATO M,SCHUSTER F,et al.Viral and murine interleukin-10 are correctly processed and retain their biological activity when produced in tobacco[J].BMC Biotechnology,2009,9(1):22.
[15]LOMONOSSOFF G P,D'AOUST M-A.Plant-produced biopharmaceuticals:a case of technical developments driving clinical deployment[J].Science,2016,353(6305):1237-1240.
[16]DAN Y.Biological functions of antioxidants in plant transformation[J].In Vitro Cellular&Developmental BiologyPlant,2008,44(3):149-161.
[17]LI S,CONG Y,LIU Y,et al.Optimization of Agrobacterium-Mediated Transformation in Soybean[J].Frontiers in Plant Science,2017(24):246-254.
[18]WROBLEWSKI T,TOMCZAK A,MICHELMORE R.Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce,tomato and Arabidopsis[J].Plant Biotechnology Journal,2005,3(2):259-273.
[19]KOMAROVA T V,BASCHIERI S,DONINI M,et al.Transient expression systems for plant-derived biopharmaceuticals[J].Expert Review of Vaccines,2010,9(8):859-876.
[20]HAHN S,GIRITCH A,BARTELS D,et al.A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants[J].Plant Biotechnology Journal,2015,13(5):708-716.
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