人源抗菌肽LL-37真核表达载体的构建及其在奶牛乳腺上皮细胞中的表达
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摘要
【目的】使人源抗菌肽LL-37在真核表达载体中获得高效表达.【方法】采用RT-PCR技术,从人的血样中扩增出LL-37基因片段,并将其连接至pcDNA3.1-His-V5(+)载体.通过脂质体介导法将重组质粒转入奶牛乳腺上皮细胞,G418筛选获得稳定阳性细胞株,并且利用RT-PCR和Western-blot检测目的基因在转录及翻译水平的表达情况.【结果】RT-PCR结果显示,经转染的乳腺上皮细胞能够扩增出523bp的LL-37基因.qRT-PCR结果发现,LL-37mRNA在转染后的不同时期均有表达,经稳定转染的表达量最高.通过Western-blot检测,获得了约17kU的目的蛋白.【结论】结果表明,LL-37基因成功整合至奶牛乳腺上皮细胞并能够正确表达,这为构建分泌LL-37蛋白的奶牛乳腺生物反应器提供了依据.
【Objective】To construct pcDNA3.1-LL-37 eukaryotic expression vector and get its high expression.【Method】The LL-37 gene amplified from human blood sample by RT-PCR was linked to the pcDNA3.1 vector.The recombinant plasmid defined as pcDNA3.1-LL-37 was testified by single XhoⅠrestriction enzyme digestion.Then the recombinant plasmid was transferred into the bovine mammary epithelial cells with liposome and was screened by G418.RT-PCR and Western-blot were applied to check the expressions of the plasmid at transcription and translation levels.【Result】The 523 bp of LL-37 gene was obtained from the post-transfection cells by RT-PCR.The mRNA expression of LL-37 gene was found in different period by qRT-PCR,the highest expression level was found at stable transfection while 17 kU specific band was obtained by Western-blot.【Conclusion】Results showed that the LL-37 gene was successful-ly inserted into the chromosome of bovine mammary epithelial cells,which provided the basis for the construction of LL-37 protein secretion to cow mammary gland bioreactor.
【Objective】To construct pcDNA3.1-LL-37 eukaryotic expression vector and get its high expression.【Method】The LL-37 gene amplified from human blood sample by RT-PCR was linked to the pcDNA3.1 vector.The recombinant plasmid defined as pcDNA3.1-LL-37 was testified by single XhoⅠrestriction enzyme digestion.Then the recombinant plasmid was transferred into the bovine mammary epithelial cells with liposome and was screened by G418.RT-PCR and Western-blot were applied to check the expressions of the plasmid at transcription and translation levels.【Result】The 523 bp of LL-37 gene was obtained from the post-transfection cells by RT-PCR.The mRNA expression of LL-37 gene was found in different period by qRT-PCR,the highest expression level was found at stable transfection while 17 kU specific band was obtained by Western-blot.【Conclusion】Results showed that the LL-37 gene was successful-ly inserted into the chromosome of bovine mammary epithelial cells,which provided the basis for the construction of LL-37 protein secretion to cow mammary gland bioreactor.
引文
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[13]侯振平,王文杰,刘景喜,等.抗菌肽的作用机制及在畜牧业中的应用饲料研究[J].饲料研究,2011(8):23-25.
[14]潘行正,黄正明,李永新,等.抗菌肽制剂对母猪死产率和仔猪成活率的影响[J].现代农业科技,2010(12):285-286.
[15]HANCOCK R E W,DIAMOND G.The role of cationic antimicrobial peptides in innate host defences[J].Trends in Microbiology,2000,8(9):402-410.
[16]ANDREAS E,MICHELLE D,MILKHAIL G,et al.A novel P2X7receptor activator,the human cathelicidinderived Peptide LL-37.induces IL-1 processing and release[J].免疫学杂志,2004,172(8):4987-4994.
[17]DRR U H N,SUDHEENDRA U S,RAMAMOORTHY A.LL-37,the only human member of the cathelicidin family of antimicrobial peptides[J].Biochimica Et Biophysica Acta,2006,1758(9):1408-25.
[18]COSTA J P D,COVA M,FERREIRA R,et al.Antimicrobial peptides:an alternative for innovative medicines applied[J].Microbiology&Biotechnology,2015,99(5):2023-2040.
[19]REINALDO R,LUCLIA D,MIGUEL G,et al.Escherichia coli expression and purification of LL-37fused to a family III carbohydrate-binding module from Clostridium thermocellum[J].Protein Expression&Purification,2009,71(1):1-7.
[20]MOON J Y,HENZLER-WILDMAN KA,RAMAMOORTHY A.Expression and purification of a recombinant LL-37fromEscherichia coli[J].Biochimica Et Biophysica Acta,2006,1758(9):1351-1358.
[21]白刚,贾怀杰,何小兵,等.羊口疮病毒ORF129基因重组质粒的构建及其在BHK-21细胞中的表达[J].甘肃农业大学学报,2013,48(5):8-13.
[2]TREON S P,BRANAGAN A R,LOAKIMIDIS L,et al.Long-term outcomes to fludarabine and rituximab in Waldenstrom macroglobulinemia[J].Blood,2009,113(16):3673-3678.
[3]MINO Y,NOBUYUKI F,YUICHI K,et al.Human cathelicidin CAP18/LL-37changes mast cell function toward innate immunity[J].Biological&Pharmaceutical Bulletin,2008,31(2):212-216.
[4]JEROLD G Y,HUANG L C,ROMANOWSKI E G,et al.Human cathelicidin(LL-37),a multifunctional peptide,is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity[J].Current Eye Research,2005,30(5):385-394.
[5]NAGAOKA I,HIROTA S,YOMOGIDA S,et al.Synergistic actions of antibacterial neutrophil defensins and cathelicidins[J].Inflammation Research,2000,49(2):73-79.
[6]BARLOW P G,LI Y,WILKINSON T S,et al.The human cationic host defense peptide LL-37mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system[J].Journal of Leukocyte Biology,2006,80(3):509-520.
[7]张蔚,孙丰南,费建文,等.内原性抗菌肽CAP18/LL37的研究进展[J].国际呼吸杂志,2006,26(9):710-712.
[8]杨永宗.中国动脉粥样硬化病理生理学研究近况[J].中国动脉硬化杂志,2004,12(4):481-489.
[9]HARADA A,SEKIDO N,AKAHOSHI T,et al.Essential involvement of interleukin-8(IL-8)in acute inflammation[J].Journal of Leukocyte Biology,1994,56(5):559-564.
[10]ULRIC H H M S,ATIYE T,SORAYA V,et al.化学增活素Gro-a(CXCL-1)、IL-8(CXCL-8)和MCP-1(CCL-2)在人体炎症角膜上的高表达[J].美国医学会眼科杂志(中文版),2004(1):58-59.
[11]BOWDISH D M E,DAVIDSON D J,ELAINE LY,et al.Impact of LL-37on anti-infective immunity[J].Journal of Leukocyte Biology,2005,77(4):451-459.
[12]汪以真,王中强,许梓荣,等.抗菌肽与抗生素的体外抗菌效果比较[J].中国兽医学报,2004,24(3):270-273.
[13]侯振平,王文杰,刘景喜,等.抗菌肽的作用机制及在畜牧业中的应用饲料研究[J].饲料研究,2011(8):23-25.
[14]潘行正,黄正明,李永新,等.抗菌肽制剂对母猪死产率和仔猪成活率的影响[J].现代农业科技,2010(12):285-286.
[15]HANCOCK R E W,DIAMOND G.The role of cationic antimicrobial peptides in innate host defences[J].Trends in Microbiology,2000,8(9):402-410.
[16]ANDREAS E,MICHELLE D,MILKHAIL G,et al.A novel P2X7receptor activator,the human cathelicidinderived Peptide LL-37.induces IL-1 processing and release[J].免疫学杂志,2004,172(8):4987-4994.
[17]DRR U H N,SUDHEENDRA U S,RAMAMOORTHY A.LL-37,the only human member of the cathelicidin family of antimicrobial peptides[J].Biochimica Et Biophysica Acta,2006,1758(9):1408-25.
[18]COSTA J P D,COVA M,FERREIRA R,et al.Antimicrobial peptides:an alternative for innovative medicines applied[J].Microbiology&Biotechnology,2015,99(5):2023-2040.
[19]REINALDO R,LUCLIA D,MIGUEL G,et al.Escherichia coli expression and purification of LL-37fused to a family III carbohydrate-binding module from Clostridium thermocellum[J].Protein Expression&Purification,2009,71(1):1-7.
[20]MOON J Y,HENZLER-WILDMAN KA,RAMAMOORTHY A.Expression and purification of a recombinant LL-37fromEscherichia coli[J].Biochimica Et Biophysica Acta,2006,1758(9):1351-1358.
[21]白刚,贾怀杰,何小兵,等.羊口疮病毒ORF129基因重组质粒的构建及其在BHK-21细胞中的表达[J].甘肃农业大学学报,2013,48(5):8-13.