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1 2016 5, 23(3): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J PepT1 闫潇, 杨丽萍, 郑文佳, 孙君君, 卢荣华, 聂国兴, 摘要 : (PepT1) (Cyprinus carpio L.), PCR PepT1 cdna, Rosetta,, PepT1 (Oryctolagus cuniculus), PepT1 ELISA, PepT1, PCR PepT1, 28 kd; PepT1 PepT1, ; PepT1,, PepT1, PepT1 PepT1, PepT1 : ; PepT1; ; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) (peptide transporter, PepT1) H + PepT1,,, (β ) [1] [2 5], 1992, Verri [6] (Anguilla anguilla) (brush border membrane, BBM), H + /Gly-Gly, (Oreochromis spp.) [7] (Sebastes caurinus) (Chionodraco hamatus) [8] BBM H + / Verri [9] 2003 (Danio rerio) PepT1 cdna, PepT1,,, (Misgurnus anguillicaudatus) [10] (Dicentrarchus labrax) [11] (Gadus morhua L.) [12] (Cyprinus carpio L.) [13] (Siniperca chuatsi) [14] (Ctenopharyngodon idellus) [15] [9] [12] [10] [11] PepT1, PCR, Western blot PepT1 [16], PepT1 cdna 收稿日期 : ; 修订日期 : 基金项目 : (14IRTSTHN013). 作者简介 : (1986 ),,. yanxiao1209@163.com 通信作者 :,. niegx@htu.cn

2 [17], PepT1, PepT1, PCR PepT1, PepT pmd 19-T-PepT1 (198 bp) PepT1, Primer Premier ( PepT1 F1: 5 -GCAGCCTCAG TCAAAGCA-3, PepT1 R1: 5 -ACAG CCACCAGGAGACAT-3 ) ( ) Trizol RNA, cdna cdna, PCR : 95 3 min; s, s, 72 2 min 15 s, 32 ; min, 4 PCR 1.0 % UNIQ-10 / (, ) DNA pgem-t (PROMEGA, ), E.coli JM109 PCR, PepT1, (BepiPred 1.0), (393~458 aa), PepT1 (198 bp), : 5 -ACAGTCCCCAACTTCCCATCAAGCTCT CAAACCCAGGTGAAGTTCCTGAACTTGGAGA ACACACCAGTGCCTGTTGTAGTGGAGGGGCA AGAACCGTTTGTTGTTCCTGGTTTTAATTCCT CTAATAATTATATGACACTGGACACTGAGAAC GTTACGGTCTCTGCTGGAGGAAGAGACGCTA CAGCTTACTTCCAG-3 Primer Premier ( BamH I EcoR I ): PepT1-F2: 5 -CGGGATCCACAGTCCCCAAC- 3 (BamH I) PepT1-R2: 5 -CGGAATTCTCACTGGAAGTA AGCTG-3 (EcoR I) BamH I EcoR I PepT1-F2 PepT1-R2, BamH I EcoR I pgem-t- PepT1, 25 µl : cdna 2.5 µl, PepT1-F1 1 µl, PepT1-R1 1 µl, dntp Mix 1 µl, 10 Taq Plus DNA Buffer 2.5 µl, Taq Plus DNA 0.3 µl, ddh 2 O 16.7 µl 94 3 min, s, s, 72 1 min, 32 ; min, 4 BamH I EcoR I PCR Amplification Kit PCR 1%, pmd 19-T, E.coli JM109, Plasmid Mini Kit I (OMEGA, ) pmd 19-T- PepT1 (198 bp) 1.2 pet-32a(+)-pept1 (198 bp) pmd 19-T-PepT1 (198 bp) pet-32a(+) BamH I EcoR I, 37, PepT1 (198 bp), E.coli Rosetta,, 1.3 pet-32a(+)-pept1 (198 bp) LB, 37, 200 r/min OD ~0.6, 1 mmol/l -β-d- (IPTG), 37, 200 r/min 8~10 h 5000 r/min 3 min,, ddh 2 O, ml Eppendorf 10 min, 5000 r/min 30 s 35 μl,

3 3 : PepT1 515 IPTG pet-32a(+), SDS-PAGE 0.3 mol/l KCl,, ddh 2 O, (1 d),, 200 mg 3 ml,,, 1.4 ( 400 µg/ml), ( 3 kg) 1 2 (SIGMA, ), 300 µg,, 3 4 (SIGMA, ), 200 µg, 4 8 d ( 1 d), 2 h, 4,,, 4 ELISA, PepT1, 1% 0.1% Tween20 PBS,, IgG- HRP (HRP conjugated), (Thermo), ( ) ( ), 450 nm : / 2.0, 1.5 PepT1 6, (80.0±0.5) g, 24 h, 6, 80, PepT1 6, 5 µm 1 %, ( DAB ) PBS Zeiss 1.6 PepT1 PCR (SYBR Premix Ex Taq TM II,, ), PrimerQuest, PepT1 (GenBank : AEX ) PepT1, β-actin (GenBank : JQ ) β-actin ( 1) Trizol 6 RNA, cdna,, PepT1-F3 PepT1-R min; s, s, 72 2 min, 33 ; min, 4 1.0%,,, PCR PepT1, 20 µl 表 1 鲤 PepT1 荧光定量引物参数 Tab. 1 Parameters of q-pcr primer pairs of PepT1 gene (5 3 ) primer sequence(5 3 ) /bp product PepT1 PepT1-F3: AATGCTGGCAGTCTTCTCTCCACT 144 PepT1-R3: TGCGATGAACACAATGAGGGCAAC 144 β-actin Actin+: GACTTCGAGCAGGAGATGG 138 Actin : CAAGAAGGATGGCTGGAACA 138,, 1.0% C T, 2 CT ( 2 CT CT 目的基因管家基因 ),

516 23 PepT1 mrna β-actin SPSS18.0 for windows one-way ANOVA LSD ± ( x ±SE) 2 2.

4 PepT1 mrna β-actin SPSS18.0 for windows one-way ANOVA LSD ± ( x ±SE) PepT1 RNA, RT-PCR PepT1 cdna,, PepT1-F PepT1-R bp ( 1) pet-32a(+), IPTG pet-32a(+)-pept1 (198 bp) Rosetta, SDS-PAGE, 28 kd ( 3) 2.4 PepT1 PepT1 OD ( 2), 1 PepT1 PCR 1, 2: PepT1 PCR ; M: 2000 bp DNA ladder. Fig. 1 Gel electrophoresis pattern of PepT1 PCR product Lane 1 and 2: PepT1 PCR product; M: 2000 bp DNA ladder. 2.2 pet-32a(+)-pept1(198 bp) PepT1 (198 bp) pmd TM 19-T Vector, pmd 19-T-PepT1 (198bp) BamH I EcoR I, pet-32a(+), pet-32a(+)-pept1 (198 bp) BamH I BamH I EcoR I ( 2), 1% 6000 bp (1 ), 6000 bp 198 bp (2 ), PepT1 pet-32a(+), pet-32a(+)-pept1 (198 bp) 2.3 IPTG pet-32a(+)-pept1 (198 bp) 2 pet-32a(+)-pept1 (198 bp) 1: pet-32a(+)-pept1(198 bp) ; 2: pet-32a(+)-pept1 (198 bp) ; M: 1 kb DNA ladder. Fig. 2 Electrophoresis analysis of pet-32a(+)-pept1 (198 bp) recombinant plasmid Lane 1: single digest of pet-32a(+)-pept1 (198 bp); Lane 2: double digest of pet-32a(+)-pept1 (198 bp); M: 1 kb DNA ladder. 3 PepT1 SDS-PAGE 1: pet32a(+) E.coli Rosetta ; 2: IPTG ; 3 4: IPTG ; M: marker. Fig. 3 SDS-PAGE pattern for the recombinant PepT1 Lane 1: E.coli Rosetta with pet32a(+); 2: E.coli Rosetta without IPTG inducement. 3, 4: E.coli Rosetta induced by IPTG; M: protein molecular weight marker (low).

5 第3期闫潇等: 鲤肠道小肽转运载体 PepT1 多克隆抗体的制备及其组织表达分析表2 Tab ELISA 测定 PepT1 抗体效价 Antibody titer of PepT1 by ELISA 序号 order number 项目 item 稀释倍数 dilution 处理组 treatment 阴性对照 negative control 空白对照 blank control 处理组/对照组 treatment/negative control 计算处理组/阴性对照组的光吸收比值, 当稀释倍数达到时, 比值为 2.78, 在稀释至倍数下, 比值为 1.71 因此, 判定 PepT1 抗血清的效价为 PepT1 在鲤不同组织中的表达分析利用制备的 PepT1 多克隆抗体, 采用免疫组织化学方法, 观察 PepT1 蛋白在鲤前肠中肠后肠肝胰脏脾和肾中的表达情况肠道组织的实验结果见图 4 阳性染色呈现棕黄色, 且从刷状缘绒毛基部到顶部逐渐加强, 主要位于中部和尖端 PepT1 蛋白在鲤前肠和中肠刷状缘上阳性染色明显, 后肠刷状缘阳性染色较浅且稀疏; 在脾肝胰脏和肾中也可观察到阳性染色见图 5 肝胰脏脾肾中阳性染色液同样呈现棕黄色, 其中, 肾的阳性颜色在近端小管刷状缘膜上有广泛分布利用荧光实时定量 PCR 检测 PepT1 在鲤各组织中的表达情况, 结果显示: PepT1 在鲤前肠中肠后肠脾肾和肝胰脏中均有表达在前肠中表达量最高 (P<0.05), 其次是中肠 (P<0.05), 在脾中表达量最少(图 6) 3 图4 鲤前肠中肠后肠 PepT1 表达的免疫组织化学检测结果 a c e 分别代表前肠中肠和后肠免疫阳性染色; b d f 分别代表前肠中肠后肠的阴性对照染色. 讨论抗体具有较高的选择性和亲和性, 可识别不同结构, 是营养生理学研究中免疫组化 Western blot 分析的一种重要工具对于高通量的抗体制 Fig. 4 Immunohistochemistry detection of PepT1 in proximal intestine, middle intestine and distal intestine of Cyprinus Carpio L. a, c, e: the positive staining in proximal intestine, middle intestine and distal intestine, respectively; b, d, f: negative control staining of proximal intestine, middle intestine and distal intestine by omitting primary antibody. 备而言, 抗原的获得是其中关键的限速步骤现个氨基酸残基组成的多肽, 免疫家兔制备抗体, 研阶段抗原的获得方法有以下几种: (1) 抗原提取究绿豆抗氰呼吸与交替氧化酶表达关系 (3) 基因 [18] 从青虾血淋巴中离心得到青虾工程抗原基因工程抗原一般分原核表达和真核血蓝蛋白, 经纯化后免疫家兔制备了多克隆抗表达, 一般是将想要表达的抗原插入表达载体(如体 (2) 人工合成的多肽主要有液相法固相 pet 表达载体)中, 导入工程菌如大肠杆菌 BL21 剂孙盛明等法和酶促合成法李驰峻等 [19] 用固相法合成由 12 等, 通过大量的表达获得大量的抗原, 韩凌霞

518 中国水产科学第 23 卷等 [20] 以重组质粒 pgem-mdgi 在大肠杆菌 BL21 中表达马利克氏病病毒特超强毒 648A 株的囊膜糖蛋白 I(gI) 完整基因, 免疫小鼠制备单克隆抗体杨晓冰等 [21] 将家蚕细胞自噬相关蛋白 Bm ATG8 的 354 bp 编码区序列连入 pet32a(+) 质粒中并转化 E.

6 518 中国水产科学第 23 卷等 [20] 以重组质粒 pgem-mdgi 在大肠杆菌 BL21 中表达马利克氏病病毒特超强毒 648A 株的囊膜糖蛋白 I(gI) 完整基因, 免疫小鼠制备单克隆抗体杨晓冰等 [21] 将家蚕细胞自噬相关蛋白 Bm ATG8 的 354 bp 编码区序列连入 pet32a(+) 质粒中并转化 E. coli Rosseta 菌株, 继而诱导表达重组 Bm ATG8 蛋白, 免疫新西兰大白兔获得多克隆抗血清本研究中采用基因工程制备抗原, 由于鲤 PepT1 属于典型的跨膜蛋白, 组织含量低且具有 12 个跨膜域, 所以不能有效地进行分离纯化, 体外表达大量具有生物活性的 PepT1 全长抗原蛋白也具有一定难度, 这成了制备 PepT1 抗体的技术瓶颈本实验在 PepT1 全长基因上选取了一段具有免疫原性长度为 198 bp 的片段, 构建到表达载体 pet-32a(+)上, 得到重组表达质粒, 通过大肠图5 鲤肝胰脏脾肾 PepT1 表达的免疫组织化学检测结果 a, c, e 分别代表肝胰脏脾肾免疫阳性染色; b, d, f 分别代表肝胰脏脾肾的阴性对照染色. Fig. 5 Immunohistochemistry detection of PepT1 in liver, spleen and kidney of Cyprinus Carpio L. a, c, e: the positive staining in liver, spleen and kidney tissues, respectively; b, d, f: negative control staining of liver, spleen and kidney tissues by omitting primary antibody. 杆菌离体表达系统体外表达出大小约为 28 kd 的融合蛋白, 成功制备了特异性强效价高的 PepT1 抗体同时, 鲤 PepT1 氨基酸序列和斑马鱼的相似性最高达 80.9%[13], 表明 PepT1 具有较高的保守性因此本实验制备的 PepT1 抗体亦可用于其他鱼类 PepT1 的表达研究同时, 本实验用包含抗原决定簇的小肽片段替代完整蛋白质抗原, 成功制备了能够识别完整蛋白的抗体, 因此, 这一基于抗原决定簇的抗体制备技术为一些难以获得足量抗原的抗体制备提供了一种可选方案, 也可以为其他功能性蛋白的性质和表达研究提供思路本研究中免疫组化染色结果显示鲤 PepT1 蛋白在前肠和中肠阳性染色较后肠明显, 且石斑鱼研究结果显示, PepT1 蛋白主要存在于肠道 BBM, 阳性染色沿着小肠绒毛从基部到顶部逐渐加强, 主要位于绒毛的中部和尖端 [22] 这与本研究结果相一致除肠道存在 PepT1 蛋白表达外, 脾肝胰脏和肾中也呈现阳性染色表明鲤肠道对小肽图6 各组织 PepT1 mrna 的相对表达量不同字母表示差异显著(P<0.05). Fig. 6 The circadian rhythm on the relative abundance PepT1 mrna in foregut, midgut, hindgut, spleen, kidney, and hepatopancreas of Cyprinus carpio L. Different letters of the same tissue indicate difference at 0.05 level among treatments. 的吸收主要集中在前肠和中肠, 提示前肠和中肠是小肽吸收的主要部位肠道是最大的消化器官, 也是蛋白代谢的主要场所, 不断地进行着蛋白质氨基酸和肽类的转运活动而在脾肝胰脏和肾中也有表达, 笔者认为这 3 种组织代谢旺盛

7 3 : PepT1 519, PepT1, PepT1 PCR PepT1, PepT1, PepT1,,, PepT1 [15] (Procypris rabaudi) [23] (Carassius auratus) [24] PepT1,, PepT1,,, PepT1, PepT1 /,, [25], PepT1, 参考文献 : [1] Rubio-Aliaga I, Daniel H. Mammalian peptide transporters as targets for drug delivery[j]. Trends Pharmacol Sci, 2002, 23(9): [2] Agar W T, Hird F J, Sidhu G S. The active absorption of amino-acids by the intestine[j]. J Physiol, 1953, 121(2): [3] Hara H, Funabiki R, Iwata M, et al. Portal absorption of small peptides in rats under unrestrained conditions[j]. J Nutr, 1984, 114(6): [4] Zhou L, Cao G T, Zhang S, et al. Intestinal fatty acid binding protein and dipeptide transporter 1 mrna in the small intestinal mucosa of weaner piglets: developmental expression and influence of glutamine[j]. China Journal of Animal Nutrition, 2012, 24(4): [,,,. 1mRNA [J]., 2012, 24(4): ] [5] Zhang A H, Zhang S, Cai H Y, et al. Regional and ontogenetic expression of PepT1, B 0 AT and EAAT3 mrna in intestine of Arbor Acre Broilers[J]. China Journal of Animal Nutrition, 2014, 26(9): [,,,. PepT1 B 0 AT EAAT3 mrna [J]., 2014, 26(9): ] [6] Verri T, Maffia M, Storelli C. H + /glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the ph-sensitive dye Acridine orange[j]. Biochim Biophys Acta (BBA), 1992, 1110: [7] Thamotharan M, Gomme J, Zonno V, et al. Electrogenic, proton- coupled, intestinal dipeptide transport in herbivorous and carnivorous teleosts[j]. Am J Physiol, 1996, 270: R [8] Maffia M, Rizzello A, Acierno R, et al. Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus[j]. J Exp Biol, 2003, 206: [9] Verri T, Kottra G, Romanoa A, et al. Molecular and functional characterisation of the zebrafish (Danio rerio) PEPT1- type peptide transporter[j]. FEBS Lett, 2003, 549: [10] Gonçalves A F, Castro L F C, Pereira-Wilson C. Is there a compromise between nutrient uptake and gas exchange in the gut of Misgurnus anguillicaudatus, an intestinal air-breathing fish?[j]. Comp Biochem Physiol D, 2007, 2(4): [11] Terova G, Corà S, Verri T, et al. Impact of feed availability on PepT1 mrna expression levels in sea bass (Dicentrarchus labrax)[j]. Aquaculture, 2009, 294: [12] Ronnestad I, Gavaia P J, Viegas C S, et al. Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects[j]. J Exp Biol, 2007, 210: [13] Ostaszewska T, Dabrowski K, Grochowski P, et al. The effect of plant protein-based diet supplemented with dipeptide or free amino acids on digestive tract morphology and PepT1 and PepT2 expressions in common carp (Cyprinus carpio L.)[J]. Comp Biochem Physiol A, 2010, 157: [14] Liu Z X, Zhang J D, Liu X Y, et al. Molecular characterization and expression research of oligopeptide transporter pept1 in Siniperca chuatsi[j]. Acta Hydrobiologica Sinica, 2014, 38(3): [,,,. PepT1 [J]., 2014, 38(3): ] [15] Feng J C, Liu Z, Lu S Q, et al. Cloning of the Pept1 gene and analysis of its expression in Ctenopharyngodon idellus tissues[j]. Journal of Fishery Sciences of China, 2013, 20(2): [,,,. PepT1 [J]., 2013, 20(2): ] [16] Romano A, Barca A, Storelli C, et al. Teleost fish models in

8 membrane transport research: the PEPT1(SLC15A1) H + -oligopeptide transporter as a case study[j]. J Physiol, 2013, 592(5): [17] Nie G X, Yan X, Wang J L, et al. Peptide transporter Pept1 in Cyprinus carpio L. s intestine: cdna cloning and sequence analysis[j]. Turk J Biochem, 2012, 37: [18] Sun S M, Ge X P, Fu H T, et al. Molecular cloning and expression analysis of the hemocyanin gene from oriental river prawn (Macrobrachium nipponense)[j]. Journal of Fishery Sciences of China, 2014, 21(2): [,,,. [J]., 2014, 21(2): ] [19] Li C J, Liang H G, Du L F, et al. Studies on the relationship between cyanide-resistant respi-ration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide[j]. Science in China: Series C, 2005, 31(1): [,,,. [J]. : C, 2005, 31(1): ] [20] Han L X, Chen L, Ding J B, et al. Preparation of monoclonal antibodies specific for MDV glycoprotein I expressed in Escherichia coli[j]. Jiangsu Agricultrual Research, 2001, 22(2): 54 57, 62.[,,,. [J]., 2001, 22(2): 54 57, 62.] [21] Yang X B, Li H J, Wu Y P, et al. Preparation of polyclonal antibody and tissue expression analysis of BmATG8 in Silkworm (Bombyx mori)[j]. Science of Sericulture, 2014, 40(5): [,,,. Bm ATG8 [J]., 2014, 40(5): ] [22] Yuen B B H, Wong C K C, Woo N Y S, et al. Induction and recovery of morpho functional changes in the intestine of juvenile carnivorous fish (Epinephelus coioides) upon exposure to foodborne benzo[a]pyrene[j]. Aquat Toxicol, 2007, 82: [23] Ye W L. PepT1 tissue expression and impact of hunger as well as soy oligopeptides on PepT1 mrna expression levels in Procypris rabaudi (Tchang)[D]. Chongqing: Chongqing Normal University, 2010: 1 60.[. PepT1 mrna [D]. :, 2010: 1 60.] [24] Xiong G. Clonging and abundance of crucian LAT2 and PePT1 gene[d]. Changsha: Hunan Agricultural University, 2009: 1 67.[. LAT2 PEPT1 [D]. :, 2009: 1 67.] [25] Xiao J H, Wu Y P, He Y, et al. Differential expression of PepT1 mrna in small intestine of Chongren Spotty Chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(2): [,,,. PepT1mRNA [J]., 2014, 45(2): ]

9 3 : PepT1 521 Preparation of the antibody and tissue distribution of the peptide transporter PepT1 in Cyprinus carpio L. YAN Xiao, YANG Liping, ZHENG Wenjia, SUN Junjun, LU Ronghua, NIE Guoxing College of Fisheries, Henan Normal University, Xinxiang , China Abstract: The lack of a PepT1 antibody for fish has hindered analysis of PepT1 protein expression by immune tissue chemistry or western blot. We analyzed the expression and distribution of PepT1 in Cyprinus carpio L. at the transcriptional and protein levels. The immunogenic cdna of PepT1 was obtained by PCR and the fragments were inserted into a pet-32a (+) Vector and transformed into Escherichia coli Rosetta. The target polypeptide was expressed after induction with 1 IPTG. The molecular weight of the recombinant protein was measured by SDS-PAGE electrophoresis. The purified PepT1 recombinant protein was used to immunize New Zealand long-eared rabbits by ear vein injection combined with subcutaneous injection for 38 d to obtain rabbit anti carp PepT1 polyclonal antibody. Enzyme-Linked Immuno Sorbent Assay (ELISA) was used to evaluate the antibody titers, immunohistochemistry was used to check the tissue expression of PepT1, and real-time fluorescent quantitative PCR was used to evaluate the expression of PepT1 at the transcriptional level. The molecular weight of the target polypeptide was ~28 kda, and the antibody titer was , suggesting that activity was high. The PepT1 protein was expressed in the foregut, midgut, hindgut, spleen, hepatopancreas, and kidneys. The level of expression was remarkably higher in the foregut and midgut than in other tissues, which may be due to their roles in absorption of peptides during digestion. The positive immune staining region in the renal tissue was obvious and clear, and consistent with short peptides being re-absorbed by PepT1 distributed on the renal tubular basement membrane. Additionally, the PepT1 transporter was also expressed in the hepatopancreas and spleen, both metabolically active tissues in carp. In conclusion, the rabbit anti-carp PepT1 polyclonal antibody prepared in this study can effectively identify PepT1 from different tissues of carp. The expression pattern of PepT1 is similar to that at the transcriptional level. Our results provide a foundation for study of the structure and function of PepT1 at both the molecular and protein levels in carp. Additionally, we provide a basis for analysis of the relationship between small peptide absorption and protein metabolism. The antibody described here will be an important tool for localization and quantitative research of PepT1 in related fish such as Carassius auratus, Ctenopharyngodon idellus, that belong to the family Cyprinidae or even in Cypriniformes. Key words: Cyprinus carpio L.; prokaryotic expression; antibody titer; tissue distribution Corresponding author: NIE Guoxing. niegx@htu.cn

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８

Microsoft Word - 3——闫 潇_news_.doc

Microsoft Word - 3——闫潇_news_.doc