中国荷斯坦奶牛MD-2基因在HEK293细胞中的表达及稳定细胞系的构建
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摘要
将中国荷斯坦奶牛髓样分化蛋白-2 (myeloid differentiation protein-2,MD-2) cDNA全长克隆至pEGFP-N1真核表达载体,利用双酶切与测序进行鉴定。经脂质体2000,将pMD-2-EGFP重组质粒转染至HEK293细胞中,应用倒置荧光显微镜、流式细胞术和Western-blot分别检测融合蛋白的表达,利用极限稀释的方法获取单细胞,并利用G418抗性筛选,获取稳定细胞系。结果发现,目的基因含有一个483 bp的开放阅读框,编码160个氨基酸。双酶切鉴定和测序结果均表明,MD-2基因编码区正确插入pEGFP-N1真核表达载体,读码框架正确,MD-2-EGFP融合蛋白在HEK293细胞中,能够成功表达;极限稀释获取单细胞,用250?g/mL的G418筛选得到的细胞系,经流式细胞术和Western-blot检测,在pMD-2-EGFP稳定细胞系组,分子质量约为46 ku处,发现了特异性条带,而pEGFP-N1空载稳定细胞组,以及空白的HEK293细胞对照组,均未发现条带。综上所述,本研究成功构建了真核表达载体pMD-2-EGFP,并在HEK293细胞中能够成功表达;经极限稀释,并利用G418抗性筛选,成功获得了pMD-2-EGFP和pEGFP-N1稳定细胞系,这为进一步研究中国荷斯坦奶牛奶牛MD-2基因的生物学功能奠定基础。
The full length of myeloid differentiation protein-2(MD-2)c DNA of the China Holstein dairy cow was cloned to the eukaryotic expression vector of p EGFP-N1,and identified by double enzyme digestion and sequenced.The recombinant plasmid p MD-2-EGFP was transfected into HEK293 cells by liposome 2000.The expression of fusion protein was detected by fluorescence microscope,flow cytometry and Western-blot.The single cell was obtained by the method of limiting dilution,and the stable cell line was obtained by using G418 resistance screening.In result,the target gene contains an open reading frame with 483 bp,encoding 160 amino acids.The double enzyme identification and sequencing results showed that the encoding region of the MD-2 gene was correctly inserted into the eukaryotic vector p EGFP-N1,the reading frame was correct,and the MD-2-EGFP fusion protein can be expressed successfully in HEK293 cells.The single cell was obtained by limit dilution,and the signle cell was screened with 250 ?g/m L G418.The flow cytometry and Western-blot detection showed that in the p MD-2-EGFP stable cell line group,the specific bands were found at the molecular weight of 46 ku,the p EGFP-N1 empty stable cell group and the blank HEK293 cell control group were not found.In conclusion,this experiment successfully constructed the eukaryotic expression vector p MD-2-EGFP,and it can be successfully expressed in HEK293 cells.Through the limiting dilution,and using G418 resistance screening,the stable cell lines of p MD-2-EGFP and p EGFPN1 were successfully obtained,which lay the foundation for further study of the biological function of the China Holstein dairy cow MD-2 gene.
The full length of myeloid differentiation protein-2(MD-2)c DNA of the China Holstein dairy cow was cloned to the eukaryotic expression vector of p EGFP-N1,and identified by double enzyme digestion and sequenced.The recombinant plasmid p MD-2-EGFP was transfected into HEK293 cells by liposome 2000.The expression of fusion protein was detected by fluorescence microscope,flow cytometry and Western-blot.The single cell was obtained by the method of limiting dilution,and the stable cell line was obtained by using G418 resistance screening.In result,the target gene contains an open reading frame with 483 bp,encoding 160 amino acids.The double enzyme identification and sequencing results showed that the encoding region of the MD-2 gene was correctly inserted into the eukaryotic vector p EGFP-N1,the reading frame was correct,and the MD-2-EGFP fusion protein can be expressed successfully in HEK293 cells.The single cell was obtained by limit dilution,and the signle cell was screened with 250 ?g/m L G418.The flow cytometry and Western-blot detection showed that in the p MD-2-EGFP stable cell line group,the specific bands were found at the molecular weight of 46 ku,the p EGFP-N1 empty stable cell group and the blank HEK293 cell control group were not found.In conclusion,this experiment successfully constructed the eukaryotic expression vector p MD-2-EGFP,and it can be successfully expressed in HEK293 cells.Through the limiting dilution,and using G418 resistance screening,the stable cell lines of p MD-2-EGFP and p EGFPN1 were successfully obtained,which lay the foundation for further study of the biological function of the China Holstein dairy cow MD-2 gene.
引文
[1] LUO M,HU L,LI D,et al.MD-2 regulates LPS-induced NLRP3 inflammasome activation and IL-1beta secretion by a MyD88/NF-κB-dependent pathway in alveolar macrophages cell line[J].Molecular Immunology,2017,90:1-10.
[2] RE F,ST ROMINGERO J L.Separate functional domains of human MD2 mediate Toll-like receptor 4 binding and lipopolysaccharide responsiveness[J].Journal of Immunology,2003,171(10):5272-5276.
[3] CANDEL S,TYRKALSKA S D,GARCIA-MORENO D,et al.Identification of evolutionarily conserved Md1 splice variants that regulate innate immunity through differential induction of NF-κB[J].Journal of Immunology,2016,15,197(4):1379-1388.
[4] OHTA S,BAHRUN U,TANAKA M,et al.Identification of a novel isoform of MD2 that downregulates lipopolysaccharide signaling[J].Biochemical and Biophysical Research Communications,2004,323(3):1103-1108.
[5] KENNEDY M N,MULLEN G E,LEIFER C A,et al.A complex of soluble MD2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4[J].Journal of Biological Chemistry,2004,279(33):34698-34704.
[6] TSUNEYOSHI N,FUKUDOME K,KOHARA J,et al.The functional and structural properties of MD2 required for lipopolysaccharide binding are absent in MD2[J].Journal of Immunology,2005,174(1):340-344.
[7] YE K,CHEN Q W,SUN Y F,et al.Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling[J].Journal of Cellular Biochemistry.2017,16,doi:10.1002/jcb.26353.
[8]焦寒伟,王洪梅,刘晓,等.中国荷斯坦奶牛髓样分化蛋白-2 cDNA的克隆与原核表达[J].中国畜牧兽医,2012,2(39):22-26.JIAO Han-wei,WANG Hong-mei,LIU Xiao,et al.Cloning and prokaryotic expression of myeloid differentiation protein-2 cDNA from Chinese Holstein cows[J].Chinese Animal Husbandry and Veterinary Medicine,2012,2(39):22-26.(in Chinese)
[9] DEY G,BHARTI R,OJHA P K,et al.Therapeutic implication of'Iturin A'for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion[J].Cell Signal,2017,35:24-36.
[10] JIN J,YU X,HU Z,et al.Isofraxidin targets the TLR4/MD-2 axis to prevent osteoarthritis development[J].Food Function,2018,doi:10.1039/c8fo01445k.
[11] HE M,BIANCHI M E,COLEMAN T R,et al.Correction to:Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance[J].Molecular Medicine,2018,24(1):31.
[12] MIYAKE K.Endotoxin recognition molecules MD2 and toll-like receptor 4 as potential targets for therapeutic intervention of endotoxin shock[J].Current Drug Targets Inflammation Allergy,2004,3(3):291-297.
[13] PUGIN J,STERN-VOEFFRAY S,DAUBEUF B,et al.Soluble MD2 activity in plasma from patient s with severe sepsis and septic shock[J].Blood,2004,104(13):4071-4079.
[14] MOBARAK E,HAVERSEN L,MANNA M,et al.Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico[J].Scientific Reports,2018,8(1):13600.
[15] COCHET F,PERI F.The Role of Carbohydrates in the lipopolysaccharide(LPS)/Toll-like receptor 4(TLR4)signalling[J].International Journal of Molecular Sciences,2017,18(11):piiE2318.
[16] XING J,LI R,LI N,et al.Anti-inflammatory effect of procyanidin B1 on LPS-treated THP1 cells via interaction with the TLR4-MD-2 heterodimer and p38MAPK and NF-κB signaling[J].Molecular and Cellular Biochemistry,2015,407(1-2):89-95.
[17] HAJJAR A M,ERNST R K,FORTUNO E S,et al.Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica[J].PLoS Pathogens,2012,8(10):e1002963.
[18] HUBER R G,BERGLUND N A,KARGAS V,et al.A thermodynamic funnel drives bacterial lipopolysaccharide transfer in the TLR4 pathway[J].Structure,2018,26(8):1151-1161.
[19] MORITA N,YAMAZAKI T,MURAKAMI Y,et al.C4b-binding protein negatively regulates TLR4/MD-2 response but not TLR3 response[J].FEBS Letter,2017,591(12):1732-1741.
[20] MATTIS D M,CHERVIN A S,RANOA D R,et al.Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses[J].Molecular Immunology,2015,68(2):203-212.
[21] WANG Y,SU L,MORIN M D,et al.TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(7):E884-93.
[2] RE F,ST ROMINGERO J L.Separate functional domains of human MD2 mediate Toll-like receptor 4 binding and lipopolysaccharide responsiveness[J].Journal of Immunology,2003,171(10):5272-5276.
[3] CANDEL S,TYRKALSKA S D,GARCIA-MORENO D,et al.Identification of evolutionarily conserved Md1 splice variants that regulate innate immunity through differential induction of NF-κB[J].Journal of Immunology,2016,15,197(4):1379-1388.
[4] OHTA S,BAHRUN U,TANAKA M,et al.Identification of a novel isoform of MD2 that downregulates lipopolysaccharide signaling[J].Biochemical and Biophysical Research Communications,2004,323(3):1103-1108.
[5] KENNEDY M N,MULLEN G E,LEIFER C A,et al.A complex of soluble MD2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4[J].Journal of Biological Chemistry,2004,279(33):34698-34704.
[6] TSUNEYOSHI N,FUKUDOME K,KOHARA J,et al.The functional and structural properties of MD2 required for lipopolysaccharide binding are absent in MD2[J].Journal of Immunology,2005,174(1):340-344.
[7] YE K,CHEN Q W,SUN Y F,et al.Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling[J].Journal of Cellular Biochemistry.2017,16,doi:10.1002/jcb.26353.
[8]焦寒伟,王洪梅,刘晓,等.中国荷斯坦奶牛髓样分化蛋白-2 cDNA的克隆与原核表达[J].中国畜牧兽医,2012,2(39):22-26.JIAO Han-wei,WANG Hong-mei,LIU Xiao,et al.Cloning and prokaryotic expression of myeloid differentiation protein-2 cDNA from Chinese Holstein cows[J].Chinese Animal Husbandry and Veterinary Medicine,2012,2(39):22-26.(in Chinese)
[9] DEY G,BHARTI R,OJHA P K,et al.Therapeutic implication of'Iturin A'for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion[J].Cell Signal,2017,35:24-36.
[10] JIN J,YU X,HU Z,et al.Isofraxidin targets the TLR4/MD-2 axis to prevent osteoarthritis development[J].Food Function,2018,doi:10.1039/c8fo01445k.
[11] HE M,BIANCHI M E,COLEMAN T R,et al.Correction to:Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance[J].Molecular Medicine,2018,24(1):31.
[12] MIYAKE K.Endotoxin recognition molecules MD2 and toll-like receptor 4 as potential targets for therapeutic intervention of endotoxin shock[J].Current Drug Targets Inflammation Allergy,2004,3(3):291-297.
[13] PUGIN J,STERN-VOEFFRAY S,DAUBEUF B,et al.Soluble MD2 activity in plasma from patient s with severe sepsis and septic shock[J].Blood,2004,104(13):4071-4079.
[14] MOBARAK E,HAVERSEN L,MANNA M,et al.Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico[J].Scientific Reports,2018,8(1):13600.
[15] COCHET F,PERI F.The Role of Carbohydrates in the lipopolysaccharide(LPS)/Toll-like receptor 4(TLR4)signalling[J].International Journal of Molecular Sciences,2017,18(11):piiE2318.
[16] XING J,LI R,LI N,et al.Anti-inflammatory effect of procyanidin B1 on LPS-treated THP1 cells via interaction with the TLR4-MD-2 heterodimer and p38MAPK and NF-κB signaling[J].Molecular and Cellular Biochemistry,2015,407(1-2):89-95.
[17] HAJJAR A M,ERNST R K,FORTUNO E S,et al.Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica[J].PLoS Pathogens,2012,8(10):e1002963.
[18] HUBER R G,BERGLUND N A,KARGAS V,et al.A thermodynamic funnel drives bacterial lipopolysaccharide transfer in the TLR4 pathway[J].Structure,2018,26(8):1151-1161.
[19] MORITA N,YAMAZAKI T,MURAKAMI Y,et al.C4b-binding protein negatively regulates TLR4/MD-2 response but not TLR3 response[J].FEBS Letter,2017,591(12):1732-1741.
[20] MATTIS D M,CHERVIN A S,RANOA D R,et al.Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses[J].Molecular Immunology,2015,68(2):203-212.
[21] WANG Y,SU L,MORIN M D,et al.TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(7):E884-93.
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