奶牛溶菌酶基因(Lyz)乳腺特异性表达质粒构建与鉴定
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摘要
研究旨在构建具有高效抗菌、活性持久的奶牛乳腺特异性表达质粒,探索研制新型抗菌制剂,为利用基因工程技术防治奶牛乳房炎提供重要材料。根据GenBank中奶牛溶菌酶基因(lysozyme,Lyz)mRNA序列(GenBank号:NM_180999.1)设计引物,从奶牛乳腺上皮细胞中RT-PCR扩增Lyz基因编码序列,将其克隆到携带增强型绿色荧光蛋白通用型表达载体pEGFP-N1中测序。重组质粒pEGFP-N1-Lyz经Ase I+Hind III双酶切除CMV启动子,将奶牛乳腺特异性表达β-乳球蛋白基因(BLG)启动子同源重组替换到Ase I和Hind III酶切位点,构建奶牛溶菌酶基因(Lyz)乳腺特异性表达质粒pBLG-EGFP-N1-Lyz,采用脂质体法转染奶牛乳腺上皮细胞(BMEC)、人肾脏上皮细胞(HEK293T),荧光显微镜检测转染细胞中绿色荧光蛋白的表达情况。结果表明,重组表达质粒pBLG-EGFP-N1-Lyz测序结果与目的片段长度相符,转染至BMEC细胞和HEK293T细胞,在奶牛乳腺上皮细胞观察到绿色荧光, HEK293T细胞未观察到绿色荧光,奶牛Lyz基因乳腺特异性表达质粒构建成功。
The purpose of this study was to construct the specific expression plasmid of dairy cow mammary gland with high efficiency and lasting activity, to explore the development of novel antibacterial agent, and to provide important materials for the prevention and treatment of dairy cow mastitis by genetic engineering technology. According to the mRNA sequence of cow Lyz gene in GenBank(NM_180999.1), a primer was designed, and the coding sequence of Lyz gene was amplified from bovine mammary epithelial cells by RT-PCR. The Lyz gene was cloned and sequenced into pEGFP-N1, a universal expression vector carrying enhanced green fluorescent protein. The recombinant plasmid pEGFP-N1-Lyz was removed from the CMV promoter by Ase I and Hind III double enzyme, and the homologous recombination of the(BLG) promoter expressing β-lactoglobulin gene was replaced by AseI and Hind III restriction sites. The(Lyz) mammary specific expression plasmid pBLG-EGFP-N1-Lyz of bovine lysozyme gene was constructed and transfected into dairy mammary epithelial cells(bMEC)and HEK293 T cells by liposome method. The expression of green fluorescent protein in transfected cells was detected by fluorescence microscope. The results showed that the pBLG-EGFP-N1-Lyz sequence of the recombinant expression plasmid was consistent with the length of the target fragment. The recombinant plasmid was transfected into bovine mammary epithelial cells and HEK293 T cells. Green fluorescence was observed in bovine mammary epithelial cells,but no green fluorescence was observed in HEK293 T cells. The mammary gland specific expression plasmid of dairy cow Lyz gene was successfully constructed.
The purpose of this study was to construct the specific expression plasmid of dairy cow mammary gland with high efficiency and lasting activity, to explore the development of novel antibacterial agent, and to provide important materials for the prevention and treatment of dairy cow mastitis by genetic engineering technology. According to the mRNA sequence of cow Lyz gene in GenBank(NM_180999.1), a primer was designed, and the coding sequence of Lyz gene was amplified from bovine mammary epithelial cells by RT-PCR. The Lyz gene was cloned and sequenced into pEGFP-N1, a universal expression vector carrying enhanced green fluorescent protein. The recombinant plasmid pEGFP-N1-Lyz was removed from the CMV promoter by Ase I and Hind III double enzyme, and the homologous recombination of the(BLG) promoter expressing β-lactoglobulin gene was replaced by AseI and Hind III restriction sites. The(Lyz) mammary specific expression plasmid pBLG-EGFP-N1-Lyz of bovine lysozyme gene was constructed and transfected into dairy mammary epithelial cells(bMEC)and HEK293 T cells by liposome method. The expression of green fluorescent protein in transfected cells was detected by fluorescence microscope. The results showed that the pBLG-EGFP-N1-Lyz sequence of the recombinant expression plasmid was consistent with the length of the target fragment. The recombinant plasmid was transfected into bovine mammary epithelial cells and HEK293 T cells. Green fluorescence was observed in bovine mammary epithelial cells,but no green fluorescence was observed in HEK293 T cells. The mammary gland specific expression plasmid of dairy cow Lyz gene was successfully constructed.
引文
[1] 李云龙,高峰,等.奶牛溶菌酶基因原核表达载体的构建[J].黑龙江畜牧兽医,2018(5):11-13.
[2] IRWIN D M,WILSON A C.Multiple cDNA sequences and the evolution of bovine stomach lysozyme[J].Biol Chem,1989,264 (19):11 387-11 393.
[3] PRIEUR D J.Tissue specific deficiency of lysozyme in ruminants[J].Comp Biochem Physiol B 1986,85(2):349-353.
[4] BENKERROUM N.Antimicrobial activity of lysozyme with specialrelevance to milk[J].African Journal of Biotechnology,2008,7 (25) :4 856-4 867.
[5] 李庆章.乳腺发育与泌乳生物学[M].北京:科学出版社,2009:346-358.
[6] SWAMINATHAN R,RAVI V K,KUMAR S,et al.Lysozyme:a model protein for amyloid research[J].Adv Protein Chem Struct Biol,2011,84 :63-111.
[7] SUGIMOTO Y,KAMADA Y,TOKUNAGA Y,et al.Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils[J].Biochem Cell Biol,2011,89(6) :533-544.
[8] SASSI P,GIUGLIARELLI A,PAOLANTONI M,et al.Unfolding and aggregation of lysozyme:a thermodynamic and kinetic study by FTIR spectroscopy[J].Biophys Chem,2011,158(1):46-53.
[9] UUSI-OUKARI M,HYTTINEN J-M,KORHONEN V-P,et al.Bovineαs1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice[J].Transgenic research,1997,6(1):75-84.
[10] BOSZE Z,AND HIRIPI L.Recombinant protein expression in milk of livestock species[J].Methods Mol Biol,2012,824(6):29-41.
[11] YANG B,WANG J,TANG B,et al.Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgeniccattle[J].PLo S One,2011,6(3):e17593.
[12] CHRENEK P,RYBAN L,VETR H,et al.Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits[J].Transgenic research,2007,16(3):353-361.
[13] MAGA E A,SHOEMAKER C F,ROWE J D,et al.Production and processing of milk from transgenic goats expressing humanl ysozyme in the mammary gland[J].J Dairy Sci,2006,89( 2) :518-524.
[14] LIU S,LI X,LU D,et al.High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC[J].Transgenic research,2012,21( 2) :407-414.
[15] 沈诚,林源,叶承荣.重组溶菌酶质粒pcDNAKLYZ 治疗泌乳期奶牛乳房炎[J].中国奶牛,2011(4) :4-6.
[2] IRWIN D M,WILSON A C.Multiple cDNA sequences and the evolution of bovine stomach lysozyme[J].Biol Chem,1989,264 (19):11 387-11 393.
[3] PRIEUR D J.Tissue specific deficiency of lysozyme in ruminants[J].Comp Biochem Physiol B 1986,85(2):349-353.
[4] BENKERROUM N.Antimicrobial activity of lysozyme with specialrelevance to milk[J].African Journal of Biotechnology,2008,7 (25) :4 856-4 867.
[5] 李庆章.乳腺发育与泌乳生物学[M].北京:科学出版社,2009:346-358.
[6] SWAMINATHAN R,RAVI V K,KUMAR S,et al.Lysozyme:a model protein for amyloid research[J].Adv Protein Chem Struct Biol,2011,84 :63-111.
[7] SUGIMOTO Y,KAMADA Y,TOKUNAGA Y,et al.Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils[J].Biochem Cell Biol,2011,89(6) :533-544.
[8] SASSI P,GIUGLIARELLI A,PAOLANTONI M,et al.Unfolding and aggregation of lysozyme:a thermodynamic and kinetic study by FTIR spectroscopy[J].Biophys Chem,2011,158(1):46-53.
[9] UUSI-OUKARI M,HYTTINEN J-M,KORHONEN V-P,et al.Bovineαs1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice[J].Transgenic research,1997,6(1):75-84.
[10] BOSZE Z,AND HIRIPI L.Recombinant protein expression in milk of livestock species[J].Methods Mol Biol,2012,824(6):29-41.
[11] YANG B,WANG J,TANG B,et al.Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgeniccattle[J].PLo S One,2011,6(3):e17593.
[12] CHRENEK P,RYBAN L,VETR H,et al.Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits[J].Transgenic research,2007,16(3):353-361.
[13] MAGA E A,SHOEMAKER C F,ROWE J D,et al.Production and processing of milk from transgenic goats expressing humanl ysozyme in the mammary gland[J].J Dairy Sci,2006,89( 2) :518-524.
[14] LIU S,LI X,LU D,et al.High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC[J].Transgenic research,2012,21( 2) :407-414.
[15] 沈诚,林源,叶承荣.重组溶菌酶质粒pcDNAKLYZ 治疗泌乳期奶牛乳房炎[J].中国奶牛,2011(4) :4-6.