SNAT2 mrna SNAT2 mr- NA 2 SNAT SNAT2 12 SNAT2 SNAT2 SNAT2 SNAT2 SNAT2 SNAT pet28a+ pbk - CMVΔ SNAT2 - HA pbk - CMV -

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1 4 2 5 Vol 42 No Journal of Shanghai Normal UniversityNatural Sciences Oct SNAT * 钠离子依赖的中性氨基酸转运蛋白 2( SNAT2) 属于 SLC38 家族, 参与小的中性氨基酸跨膜转运, 在哺乳动物组织中广泛表达 SNAT2 的功能紊乱可以导致许多神经性疾病, 如阿尔茨海默症帕金森症等采用 PCR 方法扩增得到 SNAT2 氨基端 75 个氨基酸的编码序列, 构建重组质粒 pet28a - SNAT2-75aa, 并将其转入大肠杆菌 BL21( DE3) 中, 通过 IPTG 诱导表达重组蛋白利用组氨酸标签蛋白经镍柱亲和纯化后得到了纯度在 95% 以上的 SNAT2 氨基端蛋白 SNAT2-75aa, 将其作为免疫原免疫新西兰大白兔, 从而获得 SNAT2 多克隆抗体采用巯丙基琼脂糖凝胶 6B ( Thiopropyl sepharose ) 固定抗原亲和纯化法进一步对该抗体进行纯化, 纯化后的抗体效价至少提高 10 倍蛋白免疫印迹确定该抗体对 SNAT2 具有高度的特异性, 表明 SNAT2 多克隆抗体制备的成功, 为进一步研究 SNAT2 蛋白的结构和功能奠定了生化基础转运蛋白 ; 中性氨基酸 ; 抗体 ; 蛋白纯化 Q A SLC38 SNAT 11 1 A N 1-2 A 3 SNAT1 SNAT2 SNAT SNAT2 7 SNAT1 SNAT2 SNAT MeAIB A SNAT1 SNAT SNAT4 A A N N 2 A 10 SNAT2 RNA SNAT2 mrna SNAT ZZ *

2 SNAT2 mrna SNAT2 mr- NA 2 SNAT SNAT2 12 SNAT2 SNAT2 SNAT2 SNAT2 SNAT2 SNAT pet28a+ pbk - CMVΔ SNAT2 - HA pbk - CMV - Δ - SNAT2 - HA DH5α BL21DE3 HEK293T /17 ATCC number CRL T4 DNA NEBDNA TIANGEN DMEM Gibco Lipofectamine 2000 Invitrogen Roche HA CST pbk - CMV - Δ - SNAT2 - HA PCR SNAT2 N 75 DNA 5 - AGTCGACCGGAATTCATGAAGAAGACCGAAATG - 3 EcoR1 5 - AGACCACCGCTCGAGTCACATTCCAAAGGAAGTAGT - 3 Xho1 EcoR1 Xho1 PCR pet28a T4 DH5α pet28a - SNAT2-75aa pet28a - SNAT2-75aa Kan 50 μg /mllb ~ 16 h PCR PCR 2 TaqMasterMix 5 μl 10 μmol /L 0 4 μl 1 μl 1 μl pbk - CMV - Δ - SNAT2 - HA 1 1 μl ddh 2 O ddh 2 O 10 μl PCR PCR 94 2 min s s s 72 5 min35 PCR SNAT2-75aa pet28a - SNAT2-75aa BL21DE r /min OD IPTG 0 5 mmol /L r /min 16 h SNAT2-75aa 4200 r /min 12 min 1 L 10 ml Ni Ni 25 mmol /L Tris ph mmol /L NaCl20 mmol /L Cocktail 30 μg /ml 4 30 min r /min 30 min 2 ml Ni 50 50

3 5 2SNAT ~ 24 ml mmol /L SDS - PAGE 95% SNAT2-75aa mg SNAT2-75aa 1 PBS CFA1 1 1 / mg SNAT2-75aa 1 PBS IFA SNAT2 6B Thiopropyl sepharose 1 g 15 ml 10 20mM Tris ph7 5 SNAT2 1 mg SNAT2-75aa 1 h 20 mmol /L ph 7 5 Tris Tris ph mmol /L ph 7 5 Tris 5 ~ mol /L ph 2 5 A SNAT2 SNAT2-75aa 12% SDS - PAGE SNAT2-75aa 2 58 ng 6 27 ng ng 39 2 ng 78 4 ng PVDF 1 5% TBST 1 h2 5% TBST h3 TBST 3 5 min4 5% TBST h5 TBST 3 5 min6 X pbk - CMV - Δ - SNAT2 - HA 36 h HEK293T PBS 3 RIPA 200 μl r /min 10% SDS - PAGE PVDF SNAT HA Mouse Anti - HA IgG h pet28a - SNAT2-75aa pbk - CMV - SNAT2 - HA PCR SNAT2 N 1 ~ 75 DNA 225 bp PCR pet28a+ EcoR1 Xho1 DH5α PCR 1 PCR 250 bp 3 4 SNAT2 N 75 DNA 1 PCR

4 pet28a - SNAT2-75aa SNAT2-75aa pet28a - SNAT2-75aa BL21DE3 2 L Ni SDS - PAGE SNAT2-75aa 13 kd mmol /L mmol /L 95% SNAT2-75aa mmol /L mmol /L pet28a+ - SNAT2-75aa 3 SNAT2-75aa 2 3 SNAT2 SNAT2 SNAT2-75aa 4 1 ~ 5 SNAT2-75aa 39 2 ng SNAT2 SNAT2 6B Thiopropyl sepharose SNAT2-75aa 4 6 ~ 10 SNAT ng SNAT2-75aa SNAT2 10 SNAT2 SNAT2 pbk - CMV - Δ - SNAT2 - HA HEK293T 5 1 SNAT2 2 HA SNAT2 55kD SNAT2 1 ~ 5 6 ~ 10 SNAT2-75aa 78 4ng 39 2ng 15 68ng 6 27ng 2 58ng SNAT2 SNAT2-75aa 5 1 SNAT2 2 HA HEK 293T SNAT2 3 SNAT2-13 SNAT2 SNAT2 C HA HA SNAT2 14 SNAT2 C EGFP GFP SNAT2 15 SNAT2

5 5 2SNAT2 155 SNAT2 SNAT2 SNAT2 N 75 SNAT2 3 SNAT2 6B SNAT2-75aa 10 ng SNAT2 SNAT2 HEK293 T SNAT2 SNAT2 1HAGGLUND M G SREEDHARAN S NILSSON V C et al Identification of SLC38A7 SNAT7protein as a glutamine transporter expressed in neurons J The Journal of Biological Chemistry MACKENZIE B ERICKSON J D Sodium-coupled neutral amino acid System N /Atransporters of the SLC38 gene family J Pflugers ArchivEuropean Journal of Physiology ZHANG Z GREWER C The sodium-coupled neutral amino acid transporter SNAT2 mediates an anion leak conductance that is differentially inhibited by transported substrates J Biophysical Journal ZHANG Z PAPAGEORGIOU G CORRIE J E et al Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative J Biochemistry FRANCHI-GAZZOLA R DALL'ASTA V SALA R et al The role of the neutral amino acid transporter SNAT2 in cell volume regulation J Acta Physiologica ORTIZ V ALEMAN G ESCAMILLA-DEL-ARENAL M et al Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene J American Journal of Physiology Endocrinology and Metabolism E E1102 7ZHANG Z GAMEIRO A GREWER C Highly conserved asparagine 82 controls the interaction of Na + with the sodiumcoupled neutral amino acid transporter SNAT2 J The Journal of Biological Chemistry EVANS K NASIM Z BROWN J et al Acidosis-sensing glutamine pump SNAT2 determines amino acid levels and mammalian target of rapamycin signalling to protein synthesis in L6 muscle cells J Journal of the American Society of NephrologyJASN PALII S S CHEN H KILBERG M S Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic elementj The Journal of Biological Chemistry SCHIOTH H B ROSHANBIN S HAGGLUND M G et al Evolutionary origin of amino acid transporter families SLC32 SLC36 and SLC38 and physiological pathological and therapeutic aspects J Molecular Aspects of Medicine YAO D MACKENZIE B MING H et al A novel system A isoform mediating Na + /neutral amino acid cotransport J The Journal of Biological Chemistry SHI Q PADMANABHAN R VILLEGAS C J et al Membrane topological structure of neutral system N /A amino acid transporter 4 SNAT4protein J The Journal of Biological Chemistry ZHANG Z ALBERS T FIUMERA H L et al A conserved Na+ binding site of the sodium-coupled neutral amino acid transporter 2 SNAT2 J The Journal of Biological Chemistry SNAT2 - HA J

6 SNAT2 - EGFP J Preparation of polyclonal antibody of sodium dependent neutral amino acid transporter - 2 SNAT2 LIU Li 1 MIN Weiyong 1 ZHANG Zhou 2 1 College of Life and Environment Sciences Shanghai Normal University Shanghai China 2 School of Life Science and Biopharmaceutics Shenyang Pharmaceutical University Shenyang China AbstractSodium-dependent neutral amino acid transporter 2 SNAT2 transport of small neutral amino acids is the second member of the SLC38 family and widely expresses in mammals SNAT2 dysfunction can lead to many neurological diseases such as Alzheimer disease and Parkinson's disease In order to prepare SNAT2 special antibody the gene of 75 amino acids of the N terminal of SNAT2 was amplied by PCR from pbk - CMVΔ - SNAT2 - HA plasmid The expression plasmid pet28a - SNAT2-75aa was constructed and transformed into BL21 DE3 SNAT2-75aa protein was induced to express by IPTG After purification by Ni - column the recombinant protein SNAT2-75aa was used to immunize New Zealand white rabbit in order to obtain SNAT2 polyclonal antibody In order to further purify the antibody Thiopropyl sepharose 6B column was used Western blotting results show that the purified antibody can be used specially to detect SNAT2 transient expression in HEK293 cells Successful preparation of SNAT2 polyclonal antibody will be benefit to further studying structure and function of SNAT2 Key wordstransporterneutral amino acidantibodyprotein purification ( 责任编辑 : 顾浩然 )

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<30302DB7E2C3E6CDC6BDE9A3A D32A3A92E4D4449> 绎阴元元 510515 Caco-2 Caco-2 P