人源性双亲性穿膜肽PKV14的合成及其对BHK-21细胞的影响
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摘要
目的合成人源性双亲性穿膜肽RRTLRRRRAAQRCG(PKV14),并研究其细胞穿膜效果和对细胞活性的影响。方法采用固相合成法合成PKV14,采用高效液相色谱和质谱对PKV14进行验证,使用荧光显微镜和酶标仪观察PKV14在BHK-21细胞系中的穿膜效果,采用MTT实验法检测PKV14的细胞毒性。结果合成了PKV14肽段,并且PKV14对于BHK-21细胞系的穿膜效率极高,细胞毒性实验表明PKV14对BHK-21细胞活性无影响。结论 PKV14是具有高穿膜活性,安全低毒的新型人源性细胞穿膜肽,有作为药物载体的潜力。
Objective To synthesize the human-derived amphipathic cell-penetrating peptide RRTLRRRRAAQRCG(PKV14), and investigate the effect on cell membrane penetration and its cytotoxicity. Methods PKV14 was synthesized by a solid-phase peptide synthesis. HPLC and MS analysis methods were applied to identifying its structure. The penetrating effects of PKV14 in BHK-21 cells were tested by fluorescence microscopy and microplate reader. The cytotoxicity of PKV14 was tested by MTT method. Results PKV14 was synthesized successfully and it was confirmed that PKV14 had a very high penetrating efficiency in BHK-21 cells. Cytotoxicity test showed that PKV14 had no effect on the activity of BHK-21 cells. Conclusion PKV14 is a novel human cell penetrating peptide with high penetrating activity, safety, and low toxicity which has potential as a drug carrier.
Objective To synthesize the human-derived amphipathic cell-penetrating peptide RRTLRRRRAAQRCG(PKV14), and investigate the effect on cell membrane penetration and its cytotoxicity. Methods PKV14 was synthesized by a solid-phase peptide synthesis. HPLC and MS analysis methods were applied to identifying its structure. The penetrating effects of PKV14 in BHK-21 cells were tested by fluorescence microscopy and microplate reader. The cytotoxicity of PKV14 was tested by MTT method. Results PKV14 was synthesized successfully and it was confirmed that PKV14 had a very high penetrating efficiency in BHK-21 cells. Cytotoxicity test showed that PKV14 had no effect on the activity of BHK-21 cells. Conclusion PKV14 is a novel human cell penetrating peptide with high penetrating activity, safety, and low toxicity which has potential as a drug carrier.
引文
[1]Wang F,Wang Y,Zhang X,et al.Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery[J].J Control Release,2014,174:126-136.
[2]Huang Y W,Lee H J,Tolliver L M,et al.Delivery of nucleic acid and nanomaterials by cell-penetrating peptides:opportunities and challenges[J].Biomed Res Int,2015:834079.
[3]Green M,Loewenstein P M.Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein[J].Cell,1988,55(6):1179-1188.
[4]Frankel A D,Pabo C O.Cellular uptake of the tat protein from human immunodeficiency virus[J].Cell,1988,55(6):1189-1193.
[5]Copolovici D M,Langel K,Eriste E,et al.Cellpenetrating peptides:design,synthesis,and applications[J].ACS Nano,2014,25:8(3):1972-1994.
[6]Nan Y H,Park I S,Hahm K S,et al.Antimicrobial activity,bactericidal mechanism and LPS-neutralizing activity of the cell-penetrating peptide p VEC and its analogs[J].J Pept Sci,2011,17(12):812-817.
[7]Johansson H J,El-Andaloussi S,Holm T,et al.Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein[J].Mol Ther,2008,16(1):115-123.
[8]Magzoub M,Sandgren S,Lundberg P,et al.N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis[J].Biochem Biophys Res Commun,2006,348(2):379-385.
[9]Yi Y,Kuipers O P.Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains[J].JMicrobiol Methods,2017,133:82-86.
[10]Sato M,Kosuke M,Koriyama M,et al.Timing of CRISPR/Cas9-related m RNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes[J].Theriogenology,2018,108:29-38.
[11]Henning-Knechtel A,Knechtel J,Magzoub M.DNA-assisted oligomerization of pore-forming toxin monomers into precisely-controlled protein channels[J].Nucleic Acids Res,2017,45(21):12057-12068.
[12]Eggers R,Philippi A,Altmeyer M O,et al.Primary Tcells for m RNA-mediated immunotoxin delivery[J].Gene Ther,2018,25(1):47-53.
[13]Steyer B,Carlson-Stevermer J,Angenent-Mari N,et al.High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells[J].Acta Biomater,2016,34:143-158.
[14]Di Carlo J E,Deeconda A,Tsang S H.Viral Vectors,Engineered cells and the CRISPR revolution[J].Adv Exp Med Biol.,2017,1016:3-27.
[15]Wang Q M,Gao Z,Liu S,et al.Hybrid polymeric micelles based on bioactive polypeptides as p H-responsive delivery systems against melanoma[J].Biomaterials,2014,35(25):7008-7021.
[16]Jiang Y,Li M,Zhang Z,et al.Cell-penetrating peptides as delivery enhancers for vaccine[J].Curr Pharm Biotechnol,2014,15(3):256-266.
[17]Liu S,Huang W,Jin M J,et al.High gene delivery efficiency of alkylated low-molecular-weight polyethylenimine through gemini surfactant-like effect[J].Int J Nanomed,2014.9:3567-3581.
[2]Huang Y W,Lee H J,Tolliver L M,et al.Delivery of nucleic acid and nanomaterials by cell-penetrating peptides:opportunities and challenges[J].Biomed Res Int,2015:834079.
[3]Green M,Loewenstein P M.Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein[J].Cell,1988,55(6):1179-1188.
[4]Frankel A D,Pabo C O.Cellular uptake of the tat protein from human immunodeficiency virus[J].Cell,1988,55(6):1189-1193.
[5]Copolovici D M,Langel K,Eriste E,et al.Cellpenetrating peptides:design,synthesis,and applications[J].ACS Nano,2014,25:8(3):1972-1994.
[6]Nan Y H,Park I S,Hahm K S,et al.Antimicrobial activity,bactericidal mechanism and LPS-neutralizing activity of the cell-penetrating peptide p VEC and its analogs[J].J Pept Sci,2011,17(12):812-817.
[7]Johansson H J,El-Andaloussi S,Holm T,et al.Characterization of a novel cytotoxic cell-penetrating peptide derived from p14ARF protein[J].Mol Ther,2008,16(1):115-123.
[8]Magzoub M,Sandgren S,Lundberg P,et al.N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis[J].Biochem Biophys Res Commun,2006,348(2):379-385.
[9]Yi Y,Kuipers O P.Development of an efficient electroporation method for rhizobacterial Bacillus mycoides strains[J].JMicrobiol Methods,2017,133:82-86.
[10]Sato M,Kosuke M,Koriyama M,et al.Timing of CRISPR/Cas9-related m RNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes[J].Theriogenology,2018,108:29-38.
[11]Henning-Knechtel A,Knechtel J,Magzoub M.DNA-assisted oligomerization of pore-forming toxin monomers into precisely-controlled protein channels[J].Nucleic Acids Res,2017,45(21):12057-12068.
[12]Eggers R,Philippi A,Altmeyer M O,et al.Primary Tcells for m RNA-mediated immunotoxin delivery[J].Gene Ther,2018,25(1):47-53.
[13]Steyer B,Carlson-Stevermer J,Angenent-Mari N,et al.High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells[J].Acta Biomater,2016,34:143-158.
[14]Di Carlo J E,Deeconda A,Tsang S H.Viral Vectors,Engineered cells and the CRISPR revolution[J].Adv Exp Med Biol.,2017,1016:3-27.
[15]Wang Q M,Gao Z,Liu S,et al.Hybrid polymeric micelles based on bioactive polypeptides as p H-responsive delivery systems against melanoma[J].Biomaterials,2014,35(25):7008-7021.
[16]Jiang Y,Li M,Zhang Z,et al.Cell-penetrating peptides as delivery enhancers for vaccine[J].Curr Pharm Biotechnol,2014,15(3):256-266.
[17]Liu S,Huang W,Jin M J,et al.High gene delivery efficiency of alkylated low-molecular-weight polyethylenimine through gemini surfactant-like effect[J].Int J Nanomed,2014.9:3567-3581.