版纳微型猪近交系B4GALNT2基因真核表达载体构建及亚细胞定位分析
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摘要
【目的】研究异种器官移植免疫排斥相关基因B4GALNT2的亚细胞定位情况。【方法】以版纳微型猪近交系(BMI)为试验对象,通过RT-PCR方法获得B4GALNT2基因cDNA序列,运用分子克隆技术构建B4GALNT2和报告基因EGFP的重组真核表达载体pEGFP-C1-B4GALNT2,将其转染猪肾上皮细胞PK15进行瞬时表达,同时用Mito Tracker和Hoechst33342荧光染料分别对线粒体和细胞核染色,通过EGFP示踪检测B4GALNT2在PK15细胞中的表达和定位。【结果】成功构建了BMI B4GALNT2基因的绿色荧光蛋白融合表达载体pEGFP-C1-B4GALNT2,转染PK15细胞后主要在细胞质中检测到绿色荧光蛋白的表达,试验结果与PSORTⅡserver网站的预测一致。【结论】B4GALNT2蛋白主要定位于细胞质,揭示该蛋白主要在细胞质中发挥其功能作用。
【Objective】The purpose of this study was to determine the subcellular localization of B4GALNT2 gene related immune rejection in xenotransplantation.【Method】The cDNA sequence of B4GALNT2 gene was obtained by RT-PCR using Banna minipig inbred line(BMI) as experimental animal. The recombinant eukaryotic expression vector pEGFP-C1-B4GALNT2 of the B4GALNT2 and reporter gene EGFP were constructed by molecular cloning technique. pEGFP-C1-B4GALNT2 was transfected into porcine kidney cells(PK15) for transient expression,then the nucleus and mitochondria were stained with Mito Tracker and Hoechst 33342 fluorescent dyes respectively. Finally,the expression and localization of B4GALNT2 in PK15 cells were traced by using EGFP.【Result】The green fusion expression vector pEGFP-C1-B4GALNT2 of fluorescent protein of BMI B4GALNT2 gene was successfully constructed and the expression of green fluorescent protein was detected mainly in the cytoplasm after transfecting PK15 cells,which was consistent with the prediction of PSORT Ⅱ server website.【Conclusion】B4GALNT2 protein is mainly located in the cytoplasm,revealing it plays a major role in the cytoplasm.
【Objective】The purpose of this study was to determine the subcellular localization of B4GALNT2 gene related immune rejection in xenotransplantation.【Method】The cDNA sequence of B4GALNT2 gene was obtained by RT-PCR using Banna minipig inbred line(BMI) as experimental animal. The recombinant eukaryotic expression vector pEGFP-C1-B4GALNT2 of the B4GALNT2 and reporter gene EGFP were constructed by molecular cloning technique. pEGFP-C1-B4GALNT2 was transfected into porcine kidney cells(PK15) for transient expression,then the nucleus and mitochondria were stained with Mito Tracker and Hoechst 33342 fluorescent dyes respectively. Finally,the expression and localization of B4GALNT2 in PK15 cells were traced by using EGFP.【Result】The green fusion expression vector pEGFP-C1-B4GALNT2 of fluorescent protein of BMI B4GALNT2 gene was successfully constructed and the expression of green fluorescent protein was detected mainly in the cytoplasm after transfecting PK15 cells,which was consistent with the prediction of PSORT Ⅱ server website.【Conclusion】B4GALNT2 protein is mainly located in the cytoplasm,revealing it plays a major role in the cytoplasm.
引文
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[25]LI P T,LIAO C J,YU L C,et al.Localization of B4GALNT2 and its role in mouse embryo attachment[J].Fertil Steril,2012,97(5):1206-1212.
[26]LI P T,LIAO C J,WU W G,et al.Progesterone-regulated B4galnt2expression is a requirement for embryo implantation in mice[J].Fertil Steril,2011,95(7):2404-2409.
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[28]MALAGOLINI N,SANTINI D,CHIRICOLO M,et al.Biosynthesis and expression of the Sda and sialyl Lewis x antigens in normal and cancer colon[J].Glycobiology,2007,17(7):688-697.
[29]SERAFINI-CESSI F,DALL'OLIO F.Guinea-pig kidneyβ-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein.Requirement of sialic acid in the acceptor for transferase activity[J].Biochem J,1983,215(3):483-489.
[30]PAULSON J C,WEINSTEIN J,SCHAUER A.Tissue-specific expression of sialyltransferases[J].J Biol Chem,1989,264(19):10931-10934.
[2]MATSUNARI H,NAGASHIMA H.Application of genetically modified and cloned pigs in translational research[J].J Reprod Dev,2009,55(3):225-230.
[3]REARDON S.New life for pig-to-human transplants[J].Nature,2015,527(7577):152-154.
[4]MCGREGOR C G,CARPENTIER A,LILA N,et al.Cardiac xenotransplantation technology provides materials for improved bioprosthetic heart valve[sJ].J Thorac Cardiovasc Surg,2011,141(1):269-275.
[5]COOPER D K C,GOOD A H,KOREN E,et al.Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies:relevance to discordant xenografting in man[J].Transpl Immunol,1993,1(3):198-205.
[6]DALL'OLIO F,MALAGOLINI N,CHIRICOLO M.The expanding roles of the Sd(a)_Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2[J].Biochim Biophys Acta,2014,1840(1):443-453.
[7]RAVN V,DABELSTEEN E.Tissue distribution of histo-blood group antigen[sJ].APMIS,2000,108(1):1-28.
[8]CLAUSEN H,HAKOMORI S.ABH and related histo-blood group antigens;immunochemical differences in carrier isotypes and their distribution[J].Vox Sang,1989,56(1):1-20.
[9]BIRD G W,WINGHAMJ.Cad(super Sda)in a British family with eastern connections:a note on the specificity of the Dolichos biflorus lectin[J].J Immunogenet,1976,3(5):297-302.
[10]BYRNE G W,MCGREGOR C G A,BREIMER M E.Recent investigations into pig antigen and anti-pig antibody expression[J].Int JSurg,2015,23:223-228.
[11]BYRNE G W,STALBOERGER P G,DU Z,et al.Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation[J].Transplantation,2011,91(3):287-292.
[12]BYRNE G W,STALBOERGER P G,DAVILA E,et al.Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation[J].Xenotransplantation,2008,15(4):268-276.
[13]ZHAO C,COOPER D K C,DAI Y,et al.The Sda and Cad glycan antigens and their glycosyltransferase,β1,4GalNAcT-II,in xenotransplantation[J].Xenotransplantation,2018,25(2):e12386.
[14]DROUILHET L,MANSANET C,SARRY J,et al.The highly prolific phenotype of Lacaune Sheep is associated with an ectopic expression of the B4GALNT2 gene within the ovary[J].PLoS Genet,2013,9(9):e1003809.
[15]STAUBACH F,KUNZEL S,BAINES A C,et al.Expression of the blood-grouprelated glycosyltransferase B4galnt2 influences the intestinal microbiota in mice[J].ISME J,2012,6(7):1345-1355.
[16]NGUYEN H H,JAYASINHA V,XIA B,et al.Overexpression of the cytotoxic T cell GalN Ac transferase in skeletal muscle inhibits muscular dystrophy in mdx mice[J].Proc Natl Acad Sci USA,2002,99(8):5616-5621.
[17]XU R,DEVRIES S,CAMBONI M,et al.Overexpression of Galgt2reduces dystrophic pathology in the skeletal muscles of alpha sarcoglycandeficient mice[J].Am J Pathol,2009,175(1):235-247.
[18]MONTIEL M D,KRZEWINSKI-RECCHI M A,DELANNOY P,et al.Molecular cloning,gene organization and expression of the human UDP-G a lNAc:Neu5Acα2-3Galβ-Rβ1,4-N-acetylgalactosaminyl transferase responsible for the biosynthesis of the blood group Sda/Cad antigen:evidence for an unusual extended cytoplasmic domain[J].BiochemJ,2003,373:369-379.
[19]KAWAMURA Y I,KAWASHIMA R,FUKUNAGA R,et al.Introduction of Sd(a)carbohydrate antigen in gastrointestinal cancer cells eliminates selectin ligands and inhibits metastasis[J].Cancer Res,2005,65(14):6220-6227.
[20]KLISCH K,CONTRERAS D A,SUN X,et al.The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle,pigs,horses and llama[J].Reproduction,2011,142(5):667-674.
[21]BYRNE G W,MCGREGOR C G.Cardiac xenotransplantation progress and challenges[J].Curr Opin Organ Transplant,2012,17(2):148-154.
[22]ESTRADA J L,MARTENS G,LI P,et al.Evaluation of human and nonhuman primate antibody binding to pig cells lacking GG-TA1/CMAH/beta4GalNT2 genes[J].Xenotransplantation,2015,22(3):194-202.
[23]BUTLER J R,MARTENS G R,Estrada J L,et al.Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles an alternative to direct complement regulation[J].Transgenic Res,2016,25(5):751-759.
[24]PAULSON J C,COLLEY K J.Glycosyltransferases.Structure,localization,and control of cell type-specific glycosylation[J].J Biol Chem,1989,264(30):17615-17618.
[25]LI P T,LIAO C J,YU L C,et al.Localization of B4GALNT2 and its role in mouse embryo attachment[J].Fertil Steril,2012,97(5):1206-1212.
[26]LI P T,LIAO C J,WU W G,et al.Progesterone-regulated B4galnt2expression is a requirement for embryo implantation in mice[J].Fertil Steril,2011,95(7):2404-2409.
[27]SMITH P L,LOWE J B.Molecular cloning of a murine N-acetylgalactosamine transferase cD NA that determines expression of the T lymphocyte-specific CT oligosaccharide differentiation antigen[J].J Biol Chem,1994,269(21):15162-15171.
[28]MALAGOLINI N,SANTINI D,CHIRICOLO M,et al.Biosynthesis and expression of the Sda and sialyl Lewis x antigens in normal and cancer colon[J].Glycobiology,2007,17(7):688-697.
[29]SERAFINI-CESSI F,DALL'OLIO F.Guinea-pig kidneyβ-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein.Requirement of sialic acid in the acceptor for transferase activity[J].Biochem J,1983,215(3):483-489.
[30]PAULSON J C,WEINSTEIN J,SCHAUER A.Tissue-specific expression of sialyltransferases[J].J Biol Chem,1989,264(19):10931-10934.