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Volume 43 Issue 11
Oct.  2019
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Molecular cloning, prokaryotic expression and localization analysis of C-type lectin 3 (MnLec3) cDNA from Macrobrachium nipponense

  • Corresponding author: Hongtuo FU, fuht@ffrc.cn
  • Received Date: 2018-06-15
    Accepted Date: 2018-12-04
  • C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp, a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.
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Molecular cloning, prokaryotic expression and localization analysis of C-type lectin 3 (MnLec3) cDNA from Macrobrachium nipponense

    Corresponding author: Hongtuo FU, fuht@ffrc.cn
  • 1. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Ministry of Agriculture and Rural Affairs, Wuxi    214081, China
  • 2. Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection,Yancheng Teachers University, Yancheng    224051, China
  • 3. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education Shanghai Ocean University, Shanghai    201306, China

Abstract: C-type lectins are a large family of proteins that exist in all deuterostomia. C-type lectins can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. This study aims to explore the expression patterns of C-type lectin gene in different tissues, cellular localization and becteria challenge in Macrobrachium nipponense. The cDNA sequence of M. nipponense (MnLec3) was obtained using rapid amplification of cDNA ends method (RACE) and RT-PCR. The expression levels of MnLec3 in different tissues and at different time of artificially challenged with Aeromonas hydrophilia were analyzed by qRT-PCR. The full-length cDNA sequence of MnLec3 was 1 357 bp, which contained a 5′ untranslated region of 125 bp, a 3′ untranslated region of 206 bp, a 1 026 bp open reading frame (ORF) encoding 341 amino acids. The deduced amino acid sequence of MnLec3 had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). Phylogenetic tree analysis stated that Oriental river prawn has the closest relationship with other crustacean. The expressed recombinant MnLec3 protein and polyclonal antibody were obtained in present study using a conventional method. Furthermore, immunofluorescent staining technique was used to determine cellular localization of MnLec3 in hepatopancreas of prawns. Quantitative real-time RT-PCR analysis showed that the MnLec3 gene was expressed in haemocytes, hepatopancreas, muscles, gill, testis, ovary and intestines with the highest level of expression in the hepatopancreas. Real-time PCR analysis indicated that MnLec3 transcripts level showed significant change in hepatopancreas after the prawn was artificially challenged with A. hydrophilia, followed by return to control levels at 96 h post-injection, which were similar to MnLec3 protein expression abundance using Western Blot. The results suggested that MnLec3 might be involved in the immune response against bacteria.

  • 动物细胞和植物细胞都具有合成并分泌凝集素的能力,凝集素是一种能与糖结合的蛋白质,在细胞识别和黏着反应过程中发挥重要作用[1]。C型凝集素在众多类型凝集素家族(C、P、I、F、M、L、R等)中研究得最为详细。已有学者相继报道了甲壳动物C-型凝集素的功能,包括细菌的凝集作用[2]、细胞吞噬作用[3]、细胞包被和黑化作用[4]、抗菌作用以及抗病毒活性等[5-6],其中,C型凝集素结构域家族3在甲壳动物病原识别和识别后的下游免疫反应过程中发挥重要作用[7]

    相关学者已相继克隆了多个虾蟹类C型凝集素,包括中国明对虾(Fenneropenaeus chinensis)、墨吉明对虾(F. merguiensis)、斑节对虾(Penaeus monodon)、凡纳滨对虾(Litopenaeus vannamei)、罗氏沼虾(Macrobrachium rosenbergii)、三疣梭子蟹(Portunus trituberculatus)、中华绒螯蟹(Eriocheir sinensis)等经济甲壳动物[8-14],然而,关于日本沼虾(M. nipponense) C型凝集素基因的克隆与表达分析鲜有报道。日本沼虾是我国重要的淡水养殖品种之一,由于其较高的营养价值而受到江浙沪地区消费者青睐[15-16]。近些年来,本课题组在日本沼虾人工养殖技术和群体遗传多样性方面的研究均取得了一定进展[17-20],但针对日本沼虾免疫反应机制的研究鲜有报道。日本沼虾C型凝集素结构域家族3基因(MnLec3)的研究将有助于对日本沼虾非特异性免疫系统的了解,为揭示其抗细菌感染分子机制提供有力的理论基础。

    本实验率先克隆了日本沼虾肝胰腺中C型凝集素结构域家族3基因的全长cDNA,并分析其序列特征,采用嗜水气单胞菌(Aeromonas hydrophilia)刺激后分析MnLec3基因转录水平和蛋白表达模式,通过建立MnLec3基因原核表达体系来分析其蛋白细胞定位,有助于深入研究甲壳动物的免疫应答机制。

    • 实验日本沼虾来自中国水产科学研究院淡水渔业研究中心大浦基地,在实验室暂养于循环水养殖桶2周后,挑选健康活泼的体质量为(1.28 ± 0.22) g的日本沼虾进行攻毒实验,对照组与实验组分别设置3个重复,实验组每只日本沼虾分别注射嗜水气单胞菌50 μL,细菌浓度约为2×107 CFU/mL (所用剂量依据细菌感染浓度梯度实验设定),分别在细菌刺激0、6、12、24、48 和96 h后取肝胰腺组织保存于液氮中备用。分别取日本沼虾肝胰腺、鳃、肠道、肌肉、精巢、卵巢组织及血细胞保存至−80 °C冰箱。

    • 采用酚氯仿抽提法提取RNA,采用RNA-free DNaseI纯化试剂盒纯化RNA,通过琼脂糖凝胶电泳分析和紫外分光光度计检测总RNA纯度和浓度。总RNA反转录成cDNA后保存于−80 °C冰箱,以备克隆MnLec3基因cDNA全长序列[21]。按照iScript cDNA Synthesis Kit方法获得cDNA模板来检测MnLec3基因表达水平。

    • 通过日本沼虾肝胰腺cDNA文库验证并获得C型凝集素结构域家族3编码基因序列片段,其中5′ RACE和3′ RACE的特异性扩增引物通过Primer Premiers 5.0 软件设计而成(表1),然后采用RACE技术对MnLec3编码基因5′ 端和3′ 端序列片段扩增并克隆测序分析。

      引物
      primer
      引物序列
      primer sequence
      长度/bp
      length
      引物用途
      usage
      MnLec3-GSP1 forward GGTCGGAGACCATTGCATCT 20 MnLec3 3′RACE
      MnLec3-GSP2 forward GATCATCGGAAAACTGCGGC 20
      MnLec3-GSP1 reverse GGGTGGACAGTCTCTTTGGG 20 MnLec3 5′RACE
      MnLec3-GSP2 reverse TCACCTGGGAATGGTCGTTG 20
      MnLec3 forward AAGAGGTCGGAGACCATTGC 20 MnLec3 qRT-PCR
      MnLec3 reverse CCCACCCAGTAACTTACGCC 20
      β-actin forward TATGCACTTCCTCATGCCAT 20 β-actin qRT-PCR
      β-actin reverse AGGAGGCGGCAGTGGTCAT 19

      Table 1.  Primers and their sequences used in this study

    • NCBI网站(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)的ORF Finder程序中获得日本沼虾MnLec3的开放阅读框序列,并利用BLASTP进行氨基酸序列比对,采用DNAMAN软件和MEGA 4.0软件构建系统进化树[22]

    • 采用染料法(SYBR® Premix Ex Taq Kit II,TaKaRa)在Applied Biosystems 7500荧光定量PCR仪(ABI,Carlsbad,CA,USA)上进行PCR反应。反应体积20 μL:2 μL模版,上下游引物各0.8 μL (10 μmol/L)(表1),10 μL SYBR® Premix Ex Taq II,6 μL dH2O和0.4 μL ROX Reference Dye or Dye II。反应程序:95 °C 30 s,95 °C 5 s,60 °C 34 s,共 40个循环。通过设置标准品梯度浓度进行定量PCR,分别得到MnLec3基因和内参基因的标准曲线。用双标准曲线法对实验数据进行相对定量分析[23]

    • 在嗜水气单胞菌攻毒后0、6、12、24、48和96 h时进行采样,分别从处理组和对照组中随机取3尾日本沼虾,将其肝胰腺样品保存于−80 °C冰箱,用于反转录得到cDNA模板,攻毒后MnLec3基因荧光定量分析与组织mRNA的表达分析方法相同,分别采用SPSS 17.0软件和t检验法进行多重比较及显著性分析(P<0.05为显著水平)。

    • 分别在MnLec3原核表达扩增的上游和下游引物中添加EcoRⅠ和XhoⅠ酶切位点。将扩增回收的PCR产物连接至pET28b载体,取1 μL重组的pET28b质粒转化至大肠杆菌BL21(DE3)感受态细胞中,经42 °C热击90 s,迅速转至冰上静置2 min后涂平板(30 μg/mL卡那霉素),37 °C培养过夜后进行PCR检测并送生工生物工程(上海)股份有限公司测序,序列正确的菌液内加甘油−80 °C保存备用。将培养的菌液参照本实验室原核表达反应条件大量诱导目的蛋白[21]

    • 取3只小鼠(Mus musculus),通过皮下注射蛋白抗原方法获得抗血清[生工生物工程(上海)股份有限公司],−80 °C保存备用。采用Western blot方法分析肝胰腺中凝集素C蛋白表达丰度[23],样品经SDS-PAGE后进行凝胶转膜,一抗为本实验室获得的抗血清(对照组为本实验室制备的β-actin抗体),二抗为碱性磷酸酶标记的羊抗大鼠IgG,通过BCIP/NBT碱性磷酸酶显色液显色后Bio-Rad凝胶成像仪拍照。

    • 日本沼虾肝胰腺组织于4%多聚甲醛固定液中固定24 h后,经过脱水、透明、包埋、切片等步骤,将获得的约5 µm厚度切片用于免疫荧光实验。参照已建立的日本沼虾免疫荧光方法[24],分别加入适量一抗(制备的抗血清,阴性对照一抗为未免疫大鼠血清) 于4 °C孵育过夜,用磷酸缓冲液(PBS缓冲液)冲洗3次后加入FITC标记的羊抗大鼠IgG,37 °C孵育30 min,PBS缓冲液冲洗3次后抗荧光淬灭封存液封片,荧光显微镜下观察拍照。

    2.   结果
    • MnLec3基因cDNA全长序列为1 357 bp,其中开放阅读框为1 026 bp,共编码341个氨基酸。3′端非编码区中带有Poly (A)尾及其加尾信号ATTAAA。MnLec3理论等电点(pI)约为4.40,蛋白相对分子量为38.35 ku。日本沼虾的MnLec3氨基酸序列存在17个氨基酸残基的信号肽,可确定MnLec3为分泌型蛋白。成熟肽中包含1个糖识别结构域(CRD),其中含有参与二硫键形成的6个保守的半胱氨酸,并含有2个Ca2+ 结合域,或称为糖基结合位点,是典型的C型凝集素。日本沼虾MnLec3基因cDNA序列已经提交到GenBank,登录号为KY123252。

    • 分析结果显示,日本沼虾MnLec3的氨基酸序列与罗氏沼虾C型凝集素3同源性最高。日本沼虾MnLec3氨基酸序列与凡纳滨对虾、墨吉明对虾和秀丽白虾(Exopalaemon modestus)的C型凝集素氨基酸序列进行比对分析,结果显示,MnLec3氨基酸序列中含有保守的半胱氨酸残基和糖基结合位点,这2个基序与糖基和Ca2+的结合至关重要(图1)。

      Figure 1.  Multi sequence alignment of M. nipponense C-type lectin among five species

    • 根据日本沼虾和其他动物的C型凝集素氨基酸序列构建系统进化树(图2),发现日本沼虾与甲壳动物单独聚为一支,区别于脊椎动物中鱼类、哺乳动物以及软体动物的分支,说明C型凝集素基因在进化过程中相对保守。

      Figure 2.  Phylogenetic tree based on C-type lectin amino acid sequences from other species

    • 将pET28b-MnLec3转化大肠杆菌BL21,重组菌经IPTG诱导后,其表达产物经过Ni2+亲和层析得到高纯度的目的蛋白,分子量约为36 ku (图3),与理论蛋白相对分子量相近(图4)。

      Figure 3.  Results of the MnLec3 prokaryotic expression

      Figure 4.  The results of purified rMnLec3

    • MnLec3基因在日本沼虾不同组织中的表达丰度结果显示,日本沼虾MnLec3基因在所有被检测组织中均有表达,其中肝胰腺中表达量最高,其次为血细胞、鳃、肠道、卵巢、精巢、肌肉(图5)。

      Figure 5.  qRT-PCR analysis MnLec3 mRNA expression levels in M. nipponense in different tissues

    • 攻毒后不同时间点日本沼虾肝胰腺中MnLec3基因的表达规律显示,在攻毒后6 h时,MnLec3基因表达量与对照组相比显著升高(P<0.05);随后在12和24 h时MnLec3基因表达继续升高,在48 h时达到高峰值,而在攻毒后96 h时,MnLec3基因表达量降低与对照组无显著差异(P>0.05)(图6-a)。将免疫大鼠获得的抗血清进行Western Blot检测发现,攻毒后各时间点日本沼虾肝胰腺中MnLec3蛋白的表达规律与基因转录水平基本一致,在12和48 h时MnLec3蛋白表达丰度较高,而在96 h降至与对照水平基本一致(图6-b)。

      Figure 6.  Temporal expression of MnLec3 mRNA (a) and protein abundance (b) in M. nipponense hepatopancreas injected with A. hydrophilia

    • 荧光染色结果显示,阳性信号主要位于日本沼虾肝胰腺细胞的细胞核中,细胞核中荧光染色较强;虽然肝胰腺细胞质中荧光染色相对较暗,但MnLec3蛋白在细胞质中也有少量表达(图版)。

      Figure 图版.  Distribution of MnLec3 in hepatopancreas tissue

    3.   讨论
    • 本实验获得日本沼虾MnLec3基因cDNA全长 1 357 bp,包括5′末端非翻译区125 bp、3′末端非翻译区206 bp和开放阅读框1026 bp (编码341个氨基酸)。C型凝集素是一类糖原结合蛋白家族,并在无脊椎动物中存在多个亚型[25],其需要钙离子(Ca2+)参与才能够识别碳水化合物配体。序列分析结果显示,日本沼虾MnLec3基因含有2个糖类识别结构域(CRD),已有研究报道罗氏沼虾体内的4个C型凝集素基因均含有2个CRD结构域[12],这与本实验结果一致。进一步比对氨基酸序列发现同属于长臂虾亚科的日本沼虾MnLec3与罗氏沼虾C型凝集素3氨基酸序列相似性最高(>96%)。系统发育树分析显示,日本沼虾与甲壳动物聚为一支,与其他脊椎动物、软体动物为分支,这一结果符合传统的动物系统发生分类次序。

      在虾类CRD结构域中,结合糖基分子的行为主要是通过钙离子与2个基序相互作用完成的。迄今,上述2个基序存在EPN、QPD、EPS、FRD、VND、LND、QPT、FRD等多种形式,已有学者克隆了日本沼虾C型凝集素结构域家族1基因全序列,并通过序列分析发现含有WTD和EPL 2种糖基结合位点[26],而本研究发现,日本沼虾MnLec3基因糖基结合位点分别为WHD和FDD,表明日本沼虾不同种类的C型凝集素可识别不同的糖基分子。本实验经多序列比对发现,日本沼虾C型凝集素结构域家族3基因中具有保守的半胱氨酸和色氨酸残基,揭示其具有凝血功能。本实验所克隆得到的日本沼虾MnLec3蛋白具有保守的天冬氨酸残基以及信号肽这两特征,因此推测,在结构上日本沼虾MnLec3通过吸附血淋巴中入侵的病原微生物来发挥免疫作用。本实验室成功构建了pET28b-MnLec3原核表达载体,并高效诱导表达了MnLec3重组蛋白,免疫大鼠(Rattus norvegicus)获得特异性多克隆抗体,采用制备的抗体进行免疫荧光定位分析表明MnLec3主要在肝细胞的细胞核进行表达,这与在小鼠和鱼类的研究结果相一致[27-29]

      本研究表明,日本沼虾MnLec3基因在多种组织中均有表达,其中肝胰腺中表达量最高,这与罗氏沼虾[12]和凡纳滨对虾[30]的研究结果相似。日本沼虾MnLec3基因可在精巢和卵巢中表达,这与在秀丽白虾[31]和中华绒螯蟹[14]的组织表达结果一致,表明凝集素除了执行免疫功能外,或许能够在性腺中协助生殖细胞发挥正常生理功能,但此推测尚需进一步证明。考虑到甲壳动物肝胰腺是重要物质代谢与免疫防御器官,需进一步的感染实验来验证其具体功能,笔者以日本沼虾肝胰腺为靶组织,研究其在细菌感染后MnLec3基因的表达规律。在嗜水气单胞菌刺激实验中,日本沼虾肝胰腺内MnLec3基因转录水平均出现先显著上调后下降的变化,Western blot分析也验证了这一表达规律。研究表明,该基因的表达短期内受细菌刺激诱导影响,而随着细菌在体内的大量增殖其表达反而降低,证实了凝集素在早期抵抗病菌入侵中起到模式识别受体的作用,这与日本沼虾C型凝集素结构域家族1的研究结果相一致,日本沼虾C型凝集素结构域家族1在分别受到嗜水气单胞菌和金黄色葡萄球菌(Staphylococcus awreus)刺激后其表达量同样出现显著的上调变化,日本沼虾C型凝集素结构域家族1重组蛋白已被证实对外源细菌具有凝集作用[26]。此外,中华绒螯蟹和凡纳滨对虾凝集素也参与了嗜水气单胞菌和白斑综合征病毒刺激引起的机体免疫防御反应[632-33]。由此可知,C型凝集素在甲壳动物对病原的免疫识别方面发挥至关重要的作用。

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