CN116617364A - 凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用 - Google Patents
凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域，具体的说是凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用。凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在制备病原菌的识别和凝集制剂、新型免疫制剂或饲料添加剂中的应用。本发明的重组蛋白可生产病原菌的识别制剂、菌凝集制剂、菌结合制剂等，应用于水产养殖过程中相关虾蟹疾病的治疗，或用于饲料添加剂、防腐剂或保鲜剂的生产等，另外还可以为进一步研究对虾免疫防御机制提供基础，并为凡纳滨对虾的病害防治和基因辅助选育提供参考。
Description
技术领域
本发明属于分子生物学技术领域，具体的说是凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用。
背景技术
C型凝集素是指一类含有钙离子依赖糖识别结构域(CRD)的蛋白超家族，是先天免疫中重要的模式识别受体。根据C型凝集素中CTLD的数目、排列组合方式、亚细胞定位及特殊结构等标准，脊椎动物C型凝集素可分为17个类别，其中甘露糖受体的结构比较复杂。甘露糖受体既包含胞外结构域、II型纤维连接蛋白结构域，又包含多个C-型凝集素样结构域(CRD)和跨膜结构域，可通过胞外区识别和结合特定的糖类分子，在识别病原体、递呈抗原和保持内环境稳定中发挥作用。与脊椎动物中的研究相比，无脊椎动物甘露糖受体的报道较少，亟待深入研究。
凡纳滨对虾是我国乃至全球最主要的对虾养殖品种，然而随着养殖规模的扩大，虾类疾病频发，给对虾养殖业造成了巨大损失，严重制约了对虾养殖业的健康可持续发展。对虾缺乏抗体和特异性免疫细胞，主要依靠先天免疫进行免疫防御。目前关于凡纳滨对虾C型甘露糖受体的研究尚少，其对基因的功能尚不清楚。
发明内容
本发明旨在提供一种凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用。
为实现上述目的，本发明采用的技术方案为：
一种凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在制备病原菌的识别和凝集制剂、新型免疫制剂或饲料添加剂中的应用。
所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在用于制备病原菌的识别制剂中的应用。
进一步的说对病原菌的识别，是对病原菌细胞壁保守分子即病原相关分子模式的识别和结合作用。
所述病原相关分子模式是革兰氏阴性菌的病原相关分子模式脂多糖；革兰氏阴性菌和革兰氏阳性菌的病原相关分子模式肽聚糖；真菌的病原相关分子模式葡聚糖；病毒感染相关分子模式双链RNA类似物poly(I:C)。
所述重组蛋白在制备革兰氏阴性菌的病原相关分子模式脂多糖、革兰氏阴性菌和革兰氏阳性菌的病原相关分子模式肽聚糖、真菌的病原相关分子模式葡聚糖或病毒感染相关分子模式双链RNA类似物poly(I:C)的识别制剂中的应用。
所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在用于制备革兰氏阴性菌或革兰氏阳性菌的凝集制剂中的应用。
进一步的说在10mmol L-1Ca2+存在下，重组蛋白LvCTMR对革兰氏阴性菌、革兰氏阳性菌具有明显的凝集效果。
所述革兰氏阴性菌为副溶血弧菌、溶藻弧菌或铜绿假单胞菌；革兰氏阳性菌为金黄色葡萄球菌或藤黄微球菌。
所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白含SEQ ID NO.1中所示氨基酸序列。
SEQ ID NO.1：
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEY
QGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMD
SPDLGTDDDDKAMADIGSEFRVTWNRTDLEPFPAGVEPTWNRTDLTLGS
QDCPGGYSLVGDQCLMFVNHLNLPHGEAREYCHAAEGELVAITTATDFK
NLVDYIHDSDFLARTFWVDGTDGEAEGVWVTSSGGAVPFGTPFWAAFE
YRQQPDNSAGSEHCLSLPSRWFHYMNDVLCTDAKNFICEATIQKEPEVA
SAALAPFGPSAVGVGCTLPFVEVGGLCLMFVTWAEETWHEARQVCASV
SGDLLAIADVEVLRAVYLHIQTGGLSGHTFWLGGTDVESEGTWVYTTG
EPVPMGTPFWGLSEMSAPVQEPNGGTSENCLAIGNFYNFRDYSCDSLFN
PVCVYTGKLAAALEHHHHHH
所述重组蛋白为通过PCR技术扩增编码凡纳滨对虾甘露糖受体LvMR成熟肽的基因片段并将其克隆到pET32a(+)表达载体中，在大肠杆菌BL21(DE3)实现体外重组表达，即获得含SEQ ID NO.1所示氨基酸序列的重组蛋白。
本发明所具有的优点：
本发明通过PCR技术扩增编码凡纳滨对虾甘露糖受体LvMR成熟肽的基因片段并将其克隆到pET32a(+)表达载体中，在大肠杆菌BL21(DE3)实现体外重组表达。重组蛋白LvCTMR经TALON柱纯化和透析后，对革兰氏阴性菌的病原相关分子模式脂多糖、革兰氏阴性菌和革兰氏阳性菌的病原相关分子模式肽聚糖、真菌的病原相关分子模式葡聚糖和病毒感染相关分子模式poly(I:C)均具有结合活性。重组蛋白LvCTMR对副溶血弧菌、溶藻弧菌、铜绿假单胞菌、金黄色葡萄球菌、藤黄微球菌和均有明显的凝集效果。
本发明的重组蛋白可生产病原菌的识别制剂、菌凝集制剂、菌结合制剂等，应用于水产养殖过程中相关虾蟹疾病的治疗，或用于饲料添加剂、防腐剂或保鲜剂的生产等，另外还可以为进一步研究对虾免疫防御机制提供基础，并为凡纳滨对虾的病害防治和基因辅助选育提供参考。
附图说明
图1为本发明实例提供的诱导和纯化后凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白(M：蛋白marker；1：未诱导LvCTMR菌体中表达的蛋白；2：IPTG诱导后LvCTMR表达的蛋白；3：纯化的LvCTMR重组蛋白；4：未诱导pET-32a菌体中表达的蛋白；5：IPTG诱导后pET-32a表达的蛋白；6：纯化的pET-32a重组蛋白)。
图2为本发明实例凡纳滨对虾LvCTMR重组蛋白对四种病原相关分子模式(脂多糖LPS、肽聚糖PGN、葡聚糖GLU和poly(I:C))的ELISA分析图。
图3为本发明实例凡纳滨对虾LvCTMR重组蛋白对细菌和真菌凝集活性图(检测荧光标记的副溶血弧菌、溶藻弧菌、铜绿假单胞菌、藤黄微球菌、金黄色葡萄球菌和毕赤酵母菌，rLvCTMR、rTrx、CaCl2和EDTA分别是0.2mg L-1、0.2mg L-1、10mmol L-1和10mmol L-1，Tris-HCl和rTrx分别作为空白和阴性对照)。
具体实施方式
以下结合实例对本发明的具体实施方式做进一步说明，应当指出的是，此处所描述的具体实施方式只是为了说明和解释本发明，并不局限于本发明。
实施例1.
凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的获得，具体操作如下：
根据凡纳滨对虾C型甘露糖受体LvCTMRcDNA序列，设计含有pET32a载体序列的同源臂引物LvCTMR_ReF(5'GCTGATATCGGATCCGAATTCCGAGTTACCTGGAATCGAACC3')和LvCTMR_ReR(5'CTCGAGTGCGGCCGCAAGCTTTCCAGTGTAGACACAAACTGG3')，通过PCR技术扩增编码LvCTMR成熟肽的基因片段，反应在TaKaRa PCRThermal Cycler Dice Model TP600(Takara BioInc.)中进行，反应条件为：94℃，30s；98℃，10s，58℃，30s，72℃，1min共32个循环；72℃2min。将其克隆到用用EcoR I和Hind III酶切的线性载体pET32a(+)表达载体中，转化到大肠杆菌BL21(DE3)，测序确认表达正确后，接种阳性克隆到LB培养基中，37℃振荡培养至OD600nm＝0.4～0.6，加入IPTG至终浓度为0.5mM诱导4h后离心收集菌体。菌体在冰浴条件下用超声波200W，超声2s，停2s，共超声45min。离心去掉上清，收集沉淀。沉淀以8M的尿素溶解后，利用Clontech公司的TALON柱纯化重组产物。将纯化的重组蛋白转移到透析袋中，4℃条件下，在含有2mM还原谷胱苷肽、0.2mM氧化谷胱苷肽、1mM EDTA、50mM Tris-HCl、50mMNaCl、10％甘油和1％甘氨酸的及梯度降低的尿素(6、5、4、3、2、0M)的透析复性液(pH＝8.0)中透析，使重组蛋白复性，最后在50mM Tris-HCl(pH＝8.0)的缓冲液透析2次，以除去溶液中其它成分。透析复性后的重组蛋白经PALL公司的Microsep Advance超滤离心浓缩管进行浓缩，用碧云天公司的BCA蛋白浓度测定试剂盒测得凡纳滨对虾LvCTMR重组蛋白的浓度为1.6mg/mL(参见图1)。
实施例2.
凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白PAMPs结合实验：
通过酶联免疫吸附法，检测重组蛋白对病原相关分子模式PAMPs(pathogen-associated molecular patterns，病原微生物表面某些共有的高度保守的分子结构)的结合活性。选取革兰氏阴性菌的病原相关分子模式脂多糖(lipopolysaccharide，LPS)、革兰氏阴性菌和阳性菌的病原相关分子模式肽聚糖(peptidoglycan，PGN)、真菌的病原相关分子葡聚糖(GLU)、病毒感染相关分子模式poly(I:C)为研究对象。
分别取20mg的LPS、PGN和GLU、poly(I:C)溶于100mL 50mM NaCO3-NaHCO3缓冲液(pH 9.6)。取100μL LPS、GN和GLU溶液(20μg)加入96孔酶标板，4℃包被16-18h。将96孔酶标板中未被包被的PAMPs倒出，用PBS-T清洗3次，每次5min，每孔中加入200μL PBS配制的3％BSA，37℃封闭1h。用PBS-T清洗3次，每次5min，每孔加入100μL梯度稀释的重组蛋白，同时加入5mM CaCl2和0.1mg/mL BSA，18℃孵育3h。之后，用PBS-T清洗3次，每次5min，每孔加入100μL稀释的抗His-tag的抗体(1:1000)，37℃孵育1h。然后，用PBS-T清洗3次，每次5min，每孔加入100μL稀释的HRP标记羊抗鼠的二抗(1:1000)，37℃孵育45min。用PBS-T清洗3次，每次5min，利用TMB显色试剂盒配制显色工作液，室温孵育30min，450nm波长下读数。每个实验重复3次。。结果如图2所示，重组蛋白LvMR对LPS、PGN、GLU和poly(I:C)均具有较高的结合活性，其中LvMR重组蛋白与LPS的解离常数是6.12×10-7M，与GLU的解离常数是2.39×10-4M，与poly(I:C)的解离常数是2.20×10-7M，与PGN的解离常数是1.18×10-7M。
实施例3.
凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的体外微生物凝集实验FITC染色：分别取按照常规方式培养至对数生长期的细菌、真菌1.0mL，于4℃4000rpm离心5min收集菌体，弃去培养基后用PBS洗菌3次。加入终浓度为0.1mg mL-1的FITC，暗处慢摇染色过夜。于4℃4000rpm离心5min收集菌体，弃去培养基后用大体积PBS洗菌3次，以去除残留的FITC。
其中，革兰氏阴性菌副溶血弧菌、溶藻弧菌、铜绿假单胞菌；革兰氏阳性菌金黄色葡萄球菌、藤黄微球菌；真菌毕赤酵母菌。
凝菌实验：使用无菌PBS将FITC标记的各菌重悬，调整菌浓度至1×108个mL-1。实验组中，取25μLLvCTMR重组蛋白与20μL FITC标记的菌悬液于1.5mL离心管中混匀；对照组中，取25μLrTrx与20μL FITC标记的菌悬液混匀。为观察Ca2+是否对凝集活性有影响，在实验组和对照组中分别加入10mM CaCl2和用10mM EDTA螯合Ca2+。将样品置于暗处28℃慢摇孵育2h，取5μL于荧光显微镜下观察(参见图3)。发现上述实施例LvCTMR重组蛋白在Ca2+存在下对革兰氏阴性菌副溶血弧菌、溶藻弧菌和铜绿假单胞菌、革兰氏阳性菌金黄色葡萄球菌和藤黄微球菌均有显著的凝集作用，但对真菌毕赤酵母菌没有凝集作用。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.一种凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在制备病原菌的识别和凝集制剂、新型免疫制剂或饲料添加剂中的应用。
2.按权利要求1所述的凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在用于制备病原菌的识别制剂中的应用。
3.按权利要求1所述的凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：所述重组蛋白在制备革兰氏阴性菌的病原相关分子模式脂多糖、革兰氏阴性菌和革兰氏阳性菌的病原相关分子模式肽聚糖、真菌的病原相关分子模式葡聚糖或病毒感染相关分子模式双链RNA类似物poly(I:C)的识别制剂中的应用。
4.按权利要求1所述的凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白在用于制备革兰氏阴性菌或革兰氏阳性菌的凝集制剂中的应用。
5.按权利要求4所述的凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：所述革兰氏阴性菌为副溶血弧菌、溶藻弧菌或铜绿假单胞菌；革兰氏阳性菌为金黄色葡萄球菌或藤黄微球菌。
6.按权利要求1-5任意一项所述的凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白的应用，其特征在于：所述凡纳滨对虾C型甘露糖受体LvCTMR重组蛋白含SEQ ID NO.1中所示氨基酸序列。
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