• ISSN 1000-0615
  • CN 31-1283/S
Volume 43 Issue 11
Oct.  2019
Article Contents
Turn off MathJax

Citation:

Recombinant expression and bioactivity analysis of two leptin genes of Cynoglossus semilaevis

  • Corresponding author: Yongjiang XU, xuyj@ysfri.ac.cn
  • Received Date: 2018-12-27
    Accepted Date: 2019-05-28
  • The peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis. The prokaryotic expression vector pEQ30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30, which were transformed into Escherichia coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing 6 His at the N-terminus. The obtained LepA and LepB polypeptides expressed in form of inclusion bodies had molecular weight of both 16 ku, and the optimum condition for the highest expression of the target proteins were induction by 0.5 mmol/L IPTG at 37 °C for 4 hours. The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3 mg/mL and 0.25 mg/mL, respectively. The results of Western blot and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity. The proteins were purified by Ni2+-NTA affinity chromatography, and high-purity recombinant proteins of LepA and LepB were obtained. In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from C. semilaevis indicated that they could significantly inhibit the expression of endogenous lepa, lepb and gnrh3 mRNA, which verified that the obtained recombinant proteins have biological activities. The results could help exploration of the physiological role and regulation mechanism of leptin in growth and development of C. semilaevis.
  • 加载中
  • [1] Shpilman M, Hollander-Cohen L, Ventura T, et al. Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia[J]. General and Comparative Endocrinology, 2014, 207: 74-85. doi: 10.1016/j.ygcen.2014.05.006
    [2] Kurokawa T, Uji S, Suzuki T. Identification of cDNA coding for a homologue to mammalian leptin from pufferfish, Takifugu rubripes[J]. Peptides, 2005, 26(5): 745-750. doi: 10.1016/j.peptides.2004.12.017
    [3] Kurokawa T, Murashita K. Genomic characterization of multiple leptin genes and a leptin receptor gene in the Japanese medaka, Oryzias latipes[J]. General and Comparative Endocrinology, 2009, 161(2): 229-237. doi: 10.1016/j.ygcen.2009.01.008
    [4] Gorissen M, Bernier N J, Nabuurs S B, et al. Two divergent leptin paralogues in zebrafish (Danio rerio) that originate early in teleostean evolution[J]. Journal of Endocrinology, 2009, 201(3): 329-339. doi: 10.1677/JOE-09-0034
    [5] Zhang H X, Chen H P, Zhang Y, et al. Molecular cloning, characterization and expression profiles of multiple leptin genes and a leptin receptor gene in orange-spotted grouper (Epinephelus coioides)[J]. General and Comparative Endocrinology, 2013, 181: 295-305. doi: 10.1016/j.ygcen.2012.09.008
    [6] Tang Y K, Yu J H, Li H X, et al. Molecular cloning, characterization and expression analysis of multiple leptin genes in Jian carp (Cyprinus carpio var. Jian)[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2013, 166(2): 133-140. doi: 10.1016/j.cbpb.2013.07.009
    [7] Angotzi A R, Stefansson S O, Nilsen T O, et al. Molecular cloning and genomic characterization of novel Leptin-like genes in salmonids provide new insight into the evolution of the Leptin gene family[J]. General and Comparative Endocrinology, 2013, 187: 48-59. doi: 10.1016/j.ygcen.2013.03.022
    [8] Ronnestad I, Nilsen T O, Murashita K, et al. Leptin and leptin receptor genes in Atlantic salmon: cloning, phylogeny, tissue distribution and expression correlated to long-term feeding status[J]. General and Comparative Endocrinology, 2010, 168(1): 55-70. doi: 10.1016/j.ygcen.2010.04.010
    [9] Murashita K, Uji S, Yamamoto T, et al. Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss)[J]. Comparative Biochemistry and Physiology-Part B: Biochemistry and Molecular Biology, 2008, 150(4): 377-384. doi: 10.1016/j.cbpb.2008.04.007
    [10] Frøiland E, Murashita K, Jørgensen E H, et al. Leptin and ghrelin in anadromous Arctic charr: cloning and change in expressions during a seasonal feeding cycle[J]. General and Comparative Endocrinology, 2010, 165(1): 136-143. doi: 10.1016/j.ygcen.2009.06.010
    [11] Ohga H, Matsumori K, Kodama R, et al. Two leptin genes and a leptin receptor gene of female chub mackerel (Scomber japonicus): molecular cloning, tissue distribution and expression in different obesity indices and pubertal stages[J]. General and Comparative Endocrinology, 2015, 222: 88-98. doi: 10.1016/j.ygcen.2015.06.002
    [12] Chen T, Chen S, Ren C H, et al. Two isoforms of leptin in the white-clouds mountain minnow (Tanichthys albonubes): differential regulation by estrogen despite similar response to fasting[J]. General and Comparative Endocrinology, 2016, 225: 174-184. doi: 10.1016/j.ygcen.2015.08.002
    [13] Yacobovitz M, Solomon G, Gusakovsky E E, et al. Purification and characterization of recombinant pufferfish (Takifugu rubripes) leptin[J]. General and Comparative Endocrinology, 2008, 156(1): 83-90. doi: 10.1016/j.ygcen.2007.11.013
    [14] Murashita K, Jordal A E O, Nilsen T O, et al. Leptin reduces Atlantic salmon growth through the central pro-opiomelanocortin pathway[J]. Comparative Biochemistry and Physiology-Part A: Molecular and Integrative Physiology, 2011, 158(1): 79-86. doi: 10.1016/j.cbpa.2010.09.001
    [15] Yan A F, Chen T, Chen S, et al. Goldfish leptin-AI and leptin-AII: function and central mechanism in feeding control[J]. International Journal of Molecular Sciences, 2016, 17(6): 783. doi: 10.3390/ijms17060783
    [16] Xie F F, Li X, Huang S F, et al. Identification of a soluble leptin receptor in crucian carp with different binding affinity to leptin-a and leptin-b[J]. Comparative Biochemistry and Physiology-Part B: Biochemistry and Molecular Biology, 2016, 191: 108-111. doi: 10.1016/j.cbpb.2015.10.003
    [17] 邓景耀, 孟田湘, 任胜民, 等. 渤海鱼类种类组成及数量分布[J]. 海洋水产研究, 1988(9): 10-98.Deng J Y, Meng T X, Ren S M, et al. Species composition, abundance and distribution of fishes in the Bohai sea[J]. Marine Fisheries Research, 1988(9): 10-98(in Chinese).
    [18] 柳学周, 庄志猛, 马爱军, 等. 半滑舌鳎繁殖生物学及繁育技术研究[J]. 海洋水产研究, 2005, 26(5): 7-14.Liu X Z, Zhuang Z M, Ma A J, et al. Reproductive biology and breeding technology of Cynoglossus semilaevis Günther[J]. Marine Fisheries Research, 2005, 26(5): 7-14(in Chinese).
    [19] 刘权, 王滨, 柳学周, 等. GnIH多肽对半滑舌鳎(Cynoglossus semilaevis)下丘脑生殖相关基因表达的影响[J]. 渔业科学进展, 2017, 38(1): 56-62.Liu Q, Wang B, Liu X Z, et al. Effects of gonadotropin-inhibitory hormone peptides on the reproduction-related gene expression in the hypothalamus of half-smooth tongue sole (Cynoglossus semilaevis)[J]. Progress in Fishery Sciences, 2017, 38(1): 56-62(in Chinese).
    [20] Xu Y J, Zhang Y X, Wang B, et al. Leptin and leptin receptor genes in tongue sole (Cynoglossus semilaevis): molecular cloning, tissue distribution and differential regulation of these genes by sex steroids[J]. Comparative Biochemistry and Physiology-Part A: Molecular and Integrative Physiology, 2018, 224: 11-22. doi: 10.1016/j.cbpa.2018.05.016
    [21] Chen J, Zhang Y H, Tang Z G, et al. Production of recombinant orange-spotted grouper (Epinephelus coioides) follicle-stimulating hormone (FSH) in single-chain form and dimer form by Pichia pastoris and their biological activities[J]. General and Comparative Endocrinology, 2012, 178(2): 237-249. doi: 10.1016/j.ygcen.2012.05.009
    [22] Li G G, Liang X F, Xie Q L, et al. Gene structure, recombinant expression and functional characterization of grass carp leptin[J]. General and Comparative Endocrinology, 2010, 166(1): 117-127. doi: 10.1016/j.ygcen.2009.10.009
    [23] 吴松权, 全雪丽, 朴炫春, 等. 膜荚黄芪苯丙氨酸解氨酶在大肠杆菌中的表达和纯化[J]. 延边大学农学学报, 2010, 32(4): 229-232, 253. doi: 10.3969/j.issn.1004-7999.2010.04.001Wu S Q, Quan X L, Piao X C, et al. Expression and purification of phenylalanine ammonia lyase of Astragalus membranaceus in E. coli[J]. Journal of Agricultural Science Yanbian University, 2010, 32(4): 229-232, 253(in Chinese). doi: 10.3969/j.issn.1004-7999.2010.04.001
    [24] 刘芝亮, 徐永江, 柳学周, 等. 半滑舌鳎类胰岛素生长因子-I的原核表达及活性分析[J]. 中国水产科学, 2013, 20(4): 706-712.Liu Z L, Xu Y J, Liu X Z, et al. Prokaryotic expression and bioactivity analysis of insulin-like growth factor-I from Cynoglossus semilaevis Günther[J]. Journal of Fishery Sciences of China, 2013, 20(4): 706-712(in Chinese).
    [25] 李斌, 徐永江, 柳学周, 等. 半滑舌鳎食欲素A体外重组表达及活性分析[J]. 水产学报, 2016, 40(9): 1462-1471.Li B, Xu Y J, Liu X Z, et al. Prokaryotic expression and bioactivity analysis of orexin A from Cynoglossus semilaevis[J]. Journal of Fisheries of China, 2016, 40(9): 1462-1471(in Chinese).
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(7) / Tables(1)

Article views(1708) PDF downloads(26) Cited by()

Proportional views

Recombinant expression and bioactivity analysis of two leptin genes of Cynoglossus semilaevis

    Corresponding author: Yongjiang XU, xuyj@ysfri.ac.cn
  • 1. Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
  • 2. Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao    266071, China
  • 3. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Abstract: The peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis. The prokaryotic expression vector pEQ30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30, which were transformed into Escherichia coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing 6 His at the N-terminus. The obtained LepA and LepB polypeptides expressed in form of inclusion bodies had molecular weight of both 16 ku, and the optimum condition for the highest expression of the target proteins were induction by 0.5 mmol/L IPTG at 37 °C for 4 hours. The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3 mg/mL and 0.25 mg/mL, respectively. The results of Western blot and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity. The proteins were purified by Ni2+-NTA affinity chromatography, and high-purity recombinant proteins of LepA and LepB were obtained. In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from C. semilaevis indicated that they could significantly inhibit the expression of endogenous lepa, lepb and gnrh3 mRNA, which verified that the obtained recombinant proteins have biological activities. The results could help exploration of the physiological role and regulation mechanism of leptin in growth and development of C. semilaevis.

  • 瘦素(leptin, Lep)是一种由脂肪组织分泌的蛋白质类激素,于1994年作为肥胖基因(ob)的产物首次被发现,其一般存在2种不同的分子形式(lepalepb),在摄食调控、脂肪能量代谢、生殖调控方面具有重要生理作用[1]。2005年硬骨鱼类Leptin蛋白基因首先在红鳍东方鲀(Takifugu rubripes)中被鉴定出,其主要表达部位是肝脏[2]。其后,相继在青鳉(Oryzias latipes)[3]、斑马鱼(Danio rerio)[4]、斜带石斑鱼(Epinephelus coioides)[5]、建鲤(Cyprinus carpio var. jian)[6]、大西洋鲑(Salmo salar)[7-8]、虹鳟(Oncorhynchus mykiss)[7-9]、红点鲑(Salvelinus leucomaenis)[7-10]、尼罗罗非鱼(Oreochromis niloticus)[1]、日本鲭(Scomber japonicus)[11]和唐鱼(Tanichthys albonubes)[12]等硬骨鱼类中克隆获得lepalepb基因,并对其表达特征进行了研究。为深入认识leptin的生理功能,2008年,研究人员首次成功获得重组红鳍东方鲀Leptin蛋白[13],近年来,尼罗罗非鱼[1]、虹鳟[9]、大西洋鲑[14]、金鱼(Carassius auratus)[15]和黑鲫(C. carassius)[16]等鱼类的leptin基因体外重组蛋白相继成功表达,为体外研究leptin生物学功能提供了平台和途径。

    半滑舌鳎(Cynoglossus semilaevis)是我国特有的海水养殖经济鱼类,是一种理想的近海增养殖对象[17],目前已成为我国三大鲆鲽类主导养殖品种之一。近年来,半滑舌鳎规模化人工繁育技术日趋成熟[18],已形成较大规模的养殖产业。本实验室在前期的研究中已成功克隆了半滑舌鳎leptin基因,完成了组织与时空表达特性分析。在此基础上,本研究利用原核表达载体构建了重组半滑舌鳎Leptin质粒,实现了Leptin重组蛋白的体外高效表达,同时利用下丘脑离体孵育方法验证了重组Leptin蛋白的生物活性,为从蛋白水平深入认识Leptin在半滑舌鳎生长发育中的调控机制提供了参考。

1.   材料与方法
  • 实验用半滑舌鳎取自海阳市黄海水产有限公司,3尾半滑舌鳎(全长25~30 cm、体质量350~630 g)用MS222 (300 mg/L)麻醉处死,迅速取肝脏组织保存于液氮中(−196 °C),后转入−80 °C超低温冰箱中保存,用于总RNA提取。

    30尾半滑舌鳎(体长35~40 cm、体质量550~650 g)用MS222 (300 mg/L)麻醉处死,迅速取下丘脑组织于L15液体培养基中进行离体孵育,用于Leptin重组蛋白功能的离体验证。

  • 利用RNAiso Plus (TaKaRa,日本)提取肝脏总RNA,通过超微量紫外检测仪(NanoDrop 2000 C) (Thermo,美国)测定RNA的纯度和浓度,以1%琼脂糖凝胶电泳检验RNA完整性。使用PrimeScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time)反转录试剂盒(TaKaRa,日本)合成cDNA第一链,用于leptin基因片段的扩增,操作步骤严格按照说明书进行。

  • 根据GenBank数据库中的半滑舌鳎lepa (GenBank登录号:KY432435)和lepb (GenBank登录号:MF784434)的氨基酸编码序列合成开放阅读框ORF肽段,选取BamH I和Hind III作为酶切位点。将合成得到的目的片段连接到pEASY-T1 Simple (Transgen,北京)载体上,然后导入Trans-T1 (Transgen,北京)感受态细胞中,挑选阳性克隆菌扩大培养后送至生工生物工程(上海)股份有限公司进行测序验证。

    利用质粒小提试剂盒(TaKaRa,日本)提取lepa/pEASY-T1和lepb/pEASY-T1质粒,用限制性内切酶BamH I和Hind III (TaKaRa,日本)将pQE30质粒和载有目的片段的 pEASY-T1 Simple质粒双酶切,使用T4 DNA连接酶(TaKaRa,日本)将双酶切后的目的基因和pQE30质粒表达载体连接,将连接完成的重组质粒lepa/pQE30和lepb/pQE30转化至感受态克隆菌株Top10 (Sangon Biotech,上海),筛选并扩培阳性克隆菌,然后送至生工生物工程(上海)股份有限公司进行测序验证。超微量紫外检测仪(NanoDrop 2000 C)检测重组质粒lepa/pQE30和lepb/pQE30的浓度,于15%的甘油中在−80 °C下长期保存。

  • 将测序正确的重组质粒lepa/pQE30和lepb/pQE30转化到大肠杆菌(Escherichia coli) M15 (Invitrogen,美国)中,涂布含有终浓度为50 μg/mL氨苄青霉素和30 μg/mL卡那霉素的LB固体平板上,并于37 °C、220 r/min培养过夜,然后挑取重组质粒的单菌落培养后鉴定阳性克隆。选择鉴定正确的菌液,按1∶100比例分别接种于10 mL LB培养基(含有终浓度为50 μg/mL氨苄青霉素和30 μg/mL卡那霉素)中,37 °C、220 r/min培养至OD600值达到0.6时,添加终浓度为0.5 mmol/L的IPTG,220 r/min、20 °C诱导过夜;37 °C诱导4 h,未加IPTG诱导剂的作为阴性对照。培养结束后,离心收集菌体(4 °C、4 000 r/min、10 min),PBS (pH 7.4)缓冲液洗涤并重悬菌体,以SDS-PAGE (12%分离胶)方法进行电泳检测,考马斯亮蓝染色后观察结果。

  • 收集37 °C诱导4 h的菌体沉淀和对照菌沉淀,经SDS-PAGE电泳后,利用半干电转印法将蛋白转移至PVDF膜上,用5% BSA封闭,室温下一抗和二抗分别孵育2 h,所用一抗和二抗分别为兔抗His标签(Sangon Biotech,上海)和HRP标记的羊抗兔(Sangon Biotech,上海),TMB显色,数码相机拍照。对于条带出现偏差的蛋白,交由生工生物工程(上海)股份有限公司,利用AB SCIEX TripleTOFTM 5600系统(AB,美国)进行高分辨率串联质谱鉴定分析,以验证目的蛋白结构是否正确。

  • 37 °C条件下,IPTG (0.5 mmol/L) 诱导重组菌4 h后,4 °C,12 000 r/min,离心20 min,PBS洗涤沉淀,用破碎Buffer (2 mol/L Urea,50 mmol/L Tris,300 mmol/L NaCl,0.1% Triton X-100,pH 8.0)重悬菌体,重悬后的菌液于冰浴中进行超声破碎(400 W,2 s超声,6 s间隔,20 min),破碎后用4 °C,12 000 r/min离心收集上清液。用Ni2+-NTA亲和层析柱(TaKaRa,日本)分离纯化融合蛋白,收集到的样品在透析液(1×PBS,20% Glycerol,1 mmol/L DTT,pH 8.0)中4 °C透析过夜,透析结束后用聚乙二醇20000 (PEG20000)进行浓缩,SDS-PAGE 检测。利用SK3071非干扰型蛋白定量试剂盒(Sangon Biotech,上海),按说明书操作步骤检测纯化后的重组蛋白浓度,0.22 μm滤膜过滤除菌后分装1 mL/管,−80 °C超低温冰箱保存。

  • 以L15液体培养基清洗每尾实验鱼的下丘脑组织,然后将其分别置于含10%小牛血清的L15液体培养基中培养6 h,之后在含有不同浓度Leptin重组蛋白(0、1、10、100 nmol/L)的无小牛血清的L15液体培养基中孵育,在CO2细胞培养箱(Heal Force,上海)中25 °C培养24 h,每组3个平行,离体孵育实验重复2次,培养结束后收集下丘脑组织,用于lepalepbgnrh3 mRNA的表达检测。具体离体孵育实验步骤参照本实验室刘权等[19]文章中的详细描述。

  • 离体孵育下丘脑组织提取总RNA检测Leptin蛋白基因 mRNA的表达水平变化。根据本实验室克隆得到的半滑舌鳎lepalepb和下丘脑神经肽促性腺激素释放激素gnrh3 (GenBank登录号:JQ028869)的cDNA序列分别设计定量引物,以18S rRNA (GenBank登录号:GQ426786)为内参设计定量引物18S-F和18S-R,定量PCR引物序列见表1

    引物名称
    primer name
    引物序列 (5′–3′)
    primer sequence (5′–3′)
    目的片段
    target fragment
    扩增长度/bp
    amplicon length
    lepa-RT-F CCTGGATGGTTACAACAGCCTTA lepa 223
    lepa-RT-R ATTCTTCAGCAGCACGACGAG
    lepb-RT-F GTCTGGTCGCTGCTGTTGGTT lepb 83
    lepb-RT-R TGCGTCTATGATGTGTTGGCT
    gnrh3-RT-F AGGCAGCAGAGTGATCGTG gnrh3 92
    gnrh3-RT-R CACCTGGTAGCCATCCATAAGAC
    18S-F GGTCTGTGATGCCCTTAGATGTC 18S rRNA 107
    18S-R AGTGGGGTTCAGCGGGTTAC

    Table 1.  Primers sequences used for quantitative real-time PCR

    利用Mastercycler ep realplex Real-time PCR仪(Eppendorf,德国),使用SYBR Premix Ex TaqTM II (TaKaRa,日本)试剂盒,PCR反应体系为20 μL,包括10 μL 2×SYBR® Premix Ex TaqTM II、0.8 μL上下游引物(引物浓度均为10 μmol/L)、1 μL稀释的cDNA模板以及7.4 μL无菌水,每个样品3个平行,采用两步法:95 °C 30 s;95 °C 5 s,60 °C 20 s,共40个循环,反应结束后进行熔解曲线分析。所有目的基因和参考基因的标准曲线相关系数(r2)和扩增效率(E):0.99<R2<1.00,0.9<E<1.1。以18S rRNA为内参基因,利用2−ΔΔCt的方法计算目的基因的相对表达量,结果以平均值±标准误(mean±SE) 表示。

  • 实验数据采用SPSS 22.0软件(IBM,美国)统计,对基因的mRNA表达水平进行单因素方差分析(One-Way ANOVA)与Ducan氏多重比较,统计各个基因mRNA表达水平的差异显著性。设置差异显著性水平P为0.05,当P<0.05时认为差异显著,当P<0.01时认为差异极显著。

2.   结果
  • 根据已合成的ORF肽段,经过载体双酶切和连接,构建lepa/pQE30和lepb/pQE30重组质粒(图1图2,图1括号中的数字代表该启动子或酶切位点等点位片段在质粒载体上的起始碱基数,下同)。重组蛋白N端均含有6×His标签,可进行鉴定和蛋白纯化。

    Figure 1.  Recombinant expression vector map of lepa/pQE30

    Figure 2.  Recombinant expression vector map of lepb/pQE30

  • 将半滑舌鳎lepalepb基因的重组质粒转化至大肠杆菌M15中,进行IPTG诱导表达及SDS-PAGE 电泳检测后显示,lepa/pQE30和lepb/pQE30在大肠杆菌中均有表达,在分子量16 ku处均发现一特异高表达蛋白条带,与预测的目的蛋白大小基本一致(图3图4)。经过条件探索LepA和LepB,37 °C、0.5 mmol/L IPTG诱导4 h后重组蛋白表达量最大。

    Figure 3.  SDS-PAGE analysis of lepa/pQE30 fusion protein from C. semilaevis

    Figure 4.  SDS-PAGE analysis of lepb/pQE30 fusion protein from C. semilaevis

  • 采用Western blot免疫印迹方法对37 °C下0.5 mmol/L的 IPTG诱导4 h后的LepA和LepB重组菌分别进行检测,结果显示LepA和LepB重组菌分别在PVDF膜上出现16和15 ku的单一清晰印迹(图5-a5-b),说明LepA和LepB重组菌表达的目的蛋白能被6×His抗体特异性识别,具有抗原活性,验证了半滑舌鳎LepA和LepB重组蛋白表达成功。其中LepB重组菌的Western blot检测结果与预测片段大小有一定的偏差,经质粒图谱测序,证实质粒上表达基因的氨基酸序列与半滑舌鳎lepb基因编码氨基酸序列一致,表明该蛋白为半滑舌鳎LepB重组蛋白。

    Figure 5.  Detection of recombinant LepA(a) and LepB(b) protein from C. semilaevis by Western blot

  • 取37 °C条件下0.5 mmol/L IPTG诱导4 h的LepA和LepB重组表达菌,经Ni2+-NTA亲和层析柱分离纯化目的蛋白,得到的纯化LepA和LepB重组蛋白相对分子质量均为16 ku (图6),与预期大小相符合。另外纯化后的LepA和LepB重组蛋白经蛋白定量试剂盒检测后的浓度分别为0.30和0.25 mg/mL。

    Figure 6.  SDS-PAGE for the expression of LepA and LepB fusion protein from C. semilaevis

  • 离体孵育结果表明,10 nmol/L和100 nmol/L LepA重组蛋白可显著抑制下丘脑中lepagnrh3 mRNA表达(P<0.05) (图7-a7-e),而1 nmol/L和10 nmol/L LepA重组蛋白可显著抑制下丘脑中lepb mRNA表达(P<0.05),但100 nmol/L LepA重组蛋白可上调lepb mRNA表达(P>0.05) (图7-c)。同时,外源LepB重组蛋白也可显著抑制下丘脑中lepa mRNA和lepb mRNA表达(P<0.05),但不同浓度间无显著性差异(P>0.05) (图7-b7-d)。另外,1 nmol/L和10 nmol/L LepB重组蛋白可显著抑制下丘脑中gnrh3 mRNA表达(P<0.05),但100 nmol/L LepB重组蛋白可上调gnrh3 mRNA表达(P>0.05) (图7-f)。

    Figure 7.  Effects of recombinant C. semilaevis LepA and LepB proteins on hypothalamus lepa, lepb and gnrh3 mRNA levels

3.   讨论
  • 本研究首次实现了半滑舌鳎lepalepb基因的体外原核重组表达,并纯化获得了具有生物活性的重组蛋白。已有研究证实,leptin对于鱼类的摄食、生长、生殖等生理机能都具有重要的调控作用[9, 14-15]。本实验室前期的研究表明,通过离体孵育方式,半滑舌鳎肝脏中lepalepr对性类固醇激素处理具有明显的下调表达应答,表明其在性类固醇激素的合成或调控中可能会起重要的生理作用[20]。另外,本实验室研究还发现,leptin及其受体在半滑舌鳎胚胎和仔稚幼鱼的生长发育中具有重要的调控作用。因此,通过体外重组的方式获得半滑舌鳎Leptin蛋白对于深入开展其对生殖与生长的调控作用及机制研究具有重要意义。

    本研究Western Blot结果显示,半滑舌鳎LepA和LepB重组蛋白分别在16和15 ku的位置有1条特异性结合条带,表明携带6×His标记的融合蛋白已经成功地在大肠杆菌M15中表达。但LepB重组蛋白电泳检测图谱的目的条带与理论计算值有所偏差,因此通过高分辨率串联质谱分析的方式证实了目的条带确实为半滑舌鳎LepB重组蛋白,这与Chen等[21]对斜带石斑鱼的研究结果类似,推测出现偏差的原因可能与蛋白分子量大、电泳速度慢等因素相关。而纯化后的蛋白Western Blot结果显示,半滑舌鳎LepA和LepB重组蛋白的分子量与其理论分子量16 ku基本一致,进一步证明了半滑舌鳎lepalepb基因在大肠杆菌M15中获得了成功表达,得到了半滑舌鳎LepA和LepB重组蛋白。

    以往的研究表明,鱼类的重组Leptin蛋白的生物活性都可通过体外或体内实验中生长、摄食等功能的调控作用体现。如对重组尼罗罗非鱼Leptin蛋白的研究表明,其在体外可促进已稳定转染人类LepR的BAF/3细胞(小鼠原B细胞株)增殖[1],但其效率低于哺乳动物Leptin蛋白。对草鱼(Ctenopharyngodon idella)的研究表明,皮下注射重组Leptin蛋白第1天对其摄食具有明显的抑制作用[22]。对虹鳟的研究表明,重组Leptin蛋白也会抑制其对食物的摄入,同时下调神经肽Y (NPY) mRNA表达水平[9]。另外,重组LepA蛋白通过下丘脑调节抑制金鱼食物摄入[15],并通过调控中枢促阿黑皮素原途径抑制大西洋鲑的生长[14]。本实验通过下丘脑离体孵育的方法检测了半滑舌鳎LepA和LepB重组蛋白的生物活性,在孵育液中加入半滑舌鳎LepA和LepB重组蛋白,结果显示,LepA和LepB重组蛋白均显著抑制半滑舌鳎自身lepalepb基因和gnrh3 mRNA表达水平,但100 nmol/L LepA和LepB重组蛋白可分别上调下丘脑lepbgnrh3 mRNA的表达,表明LepA和LepB重组蛋白可诱导自身leptin基因及下丘脑生殖相关基因gnrh3的表达,证明了通过原核系统体外获得的半滑舌鳎LepA和LepB重组蛋白具有明显的生物活性。下一步,将开展半滑舌鳎LepA和LepB重组蛋白的在体验证,以了解其对半滑舌鳎摄食及生长调控的生理作用及其可能机制。

    本研究选择了带有His标签的pQE30质粒作为原核表达载体,pQE表达系统是目前适应较为广泛且表达高效、操作方便的一种原核表达载体,其含有不被大肠杆菌RNA聚合酶识别的T5强启动子,低拷贝,含有6×His标签,便于重组蛋白的纯化[23]。该原核表达载体的产物为融合蛋白,为重组蛋白的纯化和检验提供了方便,且融合蛋白几乎全部以相对稳定的包涵体形式存在,避免了目的蛋白被菌体自身的蛋白酶降解,提高了目的蛋白产量[24]

    本实验利用原核表达系统获得了纯化的且具有生物活性的半滑舌鳎LepA和LepB重组蛋白,验证了LepA和LepB重组蛋白可有效调节下丘脑leptin基因的表达。原核表达系统所生产的蛋白没有真核生物的结构修饰,功能上可能会有一定的缺陷,且原核表达系统难以具有酵母表达系统的产能优势[25]。因此,在本实验结果的基础上,下一步将开展半滑舌鳎lepalepb基因的真核表达研究,获得体外制备的高活性的LepA和LepB重组蛋白,为研制应用于养殖生产的绿色高效摄食和生长代谢调剂制剂提供理论和技术支持。

Reference (25)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return