CN110358771A - 大黄鱼清道夫受体scara3基因及其用途 - Google Patents
大黄鱼清道夫受体scara3基因及其用途 Download PDFInfo
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- CN110358771A CN110358771A CN201910752882.XA CN201910752882A CN110358771A CN 110358771 A CN110358771 A CN 110358771A CN 201910752882 A CN201910752882 A CN 201910752882A CN 110358771 A CN110358771 A CN 110358771A
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- scara3
- larimichthys crocea
- scavenger receptor
- vibrio harveyi
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Abstract
本发明提供一种大黄鱼清道夫受体SCARA3基因及其用途，属于基因工程技术领域，大黄鱼清道夫受体SCARA3基因完整ORF包含1938个碱基，核苷酸序列如SEQ ID NO:1所示，在哈维弧菌的感染下，该基因表达量上调，可应用于大黄鱼亲鱼抗哈维弧菌力的检测和/或抗哈维弧菌良种的选育，尤其可以应用于大黄鱼亲鱼抗哈维弧菌力的检测和/或抗哈维弧菌良种的选育。本发明大黄鱼清道夫受体SCARA3基因编码的大黄鱼清道夫受体蛋白的重组表达蛋白具有显著的抑哈维弧菌活性，可用于制备抑制哈维弧菌的生物制品，为大黄鱼抗哈维弧菌机理研究和抗哈维弧菌饲料添加剂的研制提供基因序列和方法。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种大黄鱼清道夫受体SCARA3基因及其用途。
背景技术
大黄鱼(Larimichthys crocea)属鲈形目(Perciformes)，石首鱼科(Sciaenidae)，黄鱼属(Larimichthys)，俗称“黄鱼”、“黄瓜鱼”、“大黄花鱼”，是东海海域沿海地带最重要的鱼类养殖品种。大黄鱼体色金黄、唇部鲜红、肉质细嫩、味道鲜美且营养价值高，作为我国传统“四大海产”和传统的“国鱼”在我国及太平洋西部海洋渔业中占有重要地位。但随着养殖业的迅猛发展和养殖规模的不断扩大，病害问题日益凸显，其中由哈维弧菌引起的弧菌病是危害较为严重的细菌性疾病，被感染的鱼类因种类、体质、部位等不同而表现出复杂多样的症状，且引起较高的死亡率，已严重威胁大黄鱼的养殖，极易造成巨大的经济损失。机体通过免疫细胞表面模式识别受体识别和结合病原体相关分子模式，触发机体的先天性和适应性免疫反应。清道夫受体(Scavenger receptors,SRs)和Toll样受体以及甘露糖受体一样，都是一种重要的模式识别受体，在机体的免疫中发挥重要功能，而A型清道夫受体基因家族则是清道夫受体基因家族中最为重要的亚家族，在病原体识别，免疫应答以及内稳态等过程中都起到十分重要的作用。目前对A家族的研究也最为深入，它是一类II型跨膜受体，由N端胞内区、跨膜区、α-螺旋、胶原样结构域和C端半胱氨酸丰富区(SRCR结构域)组成。SR-A所形成的三聚体膜蛋白可以有效结合病原体模式相关分子，其中包括修饰后的LPS、多糖分子以及多聚核苷酸(polyI:C、polyG)，特别是可以有效地结合修饰后的低密度脂蛋白LDL，进而激活免疫通路产生相关的免疫作用。A型清道夫受体基因家族包括5个成员，分别是巨噬细胞清道夫受体1(SCARA1/SR-A1/MASR1)、胶原样巨噬细胞受体(SCARA2/SCARA3)、细胞应激反应蛋白(SCARA3/CSR1)、C型凝集素清道夫受体(SCARA4/CLP1/COLEC12)和A型清道夫受体5(SCARA5/TESR)。其中，清道夫受体A家族的SCARA3基因作为天然免疫系统的关键分子，可识别大量异己抗原，是机体免疫防御的重要防线。
发明内容
本发明的一个目的在于提供一种同源克隆法及RACE技术获得的大黄鱼清道夫受体SCARA3基因，在哈维弧菌的感染下，该基因表达量上调，对哈维弧菌感染后的识别较为敏感且反应迅速，最高表达是在感染后24h，感染后96h后仍有较高的表达。
本发明为实现上述目的所采取的技术方案为：
大黄鱼清道夫受体SCARA3基因，其完整ORF包含1938个碱基，核苷酸序列如SEQ IDNO:1所示。大黄鱼清道夫受体SCARA3基因和其他物种的SCARA3基因具有较高相似性，其理论分子量和等电点分别为70.94kDa和7.02，存在一个跨膜螺旋区(85-107aa)，两个α-螺旋区(147-219aa和326-354aa)以及一个胶原样结构域(519-584aa)，且存在3个Major tumornecrosis factor receptor 2(TRAF2)因子，分别是T-G-E-E(58-61aa)、S-E-E-E(116-119aa)和A-D-E-E(267-270aa)，同时还存在2个酪氨酸活化区(Y-G-F-V，91-94aa和Y-Y-D-L，323-326aa)。大黄鱼清道夫受体SCARA3基因共有13个外显子，12个内含子。
优选的，大黄鱼清道夫受体SCARA3基因完整ORF通过如下方法获得：
S1：将收集得到的各类样品进行总RNA提取；
S2：对S1提取的RNA进行反转录，获得相应的cDNA第一条链的合成；
S3：以cDNA为模板进行PCR扩增，扩增大黄鱼清道夫受体SCARA3基因，纯化；
S4：将S3纯化后产物进行T-A克隆，检测PCR产物，将含有合适片段大小的重组子进行DNA序列测序。本发明通过同源克隆法及RACE技术获得大黄鱼清道夫受体SCARA3开放阅读框序列。
更优选的，PCR扩增用引物，其上游引物的序列为SEQ ID NO:2，下游引物的序列为SEQ ID NO:3。
SCARA3-F:5'-AGAGTGACTCGAGCTCTAGCG-3'；
SCARA3-R:5'-GTATTGACTTATGGAGCCAGCATT-3'。
更优选的，大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下，基因表达量明显升高。大黄鱼清道夫受体SCARA3基因对哈维弧菌感染后的识别较为敏感且反应迅速，最高表达是在感染后24h，感染后96h后仍有较高的表达，表明在哈维弧菌的刺激下，SCARA3基因参与大黄鱼类的免疫过程。
上述大黄鱼清道夫受体SCARA3基因在筛选/检测大黄鱼抗哈维弧菌个体中的用途，大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下，基因表达量上调。尤其可以应用于大黄鱼亲鱼抗哈维弧菌力的检测和/或抗哈维弧菌良种的选育。
上述大黄鱼清道夫受体SCARA3基因的检测方法，检测大黄鱼脾脏和/或肌肉和/或皮肤和/或脑和/或肝脏中SCARA3基因的表达量的方法。
优选的，检测方法通过PCR扩增引物来扩增大黄鱼脾脏和/或肌肉和/或皮肤和/或脑和/或肝脏中的RNA。SCARA3基因在大黄鱼肝脏、脾脏、脑、心脏、头肾、肌肉、皮肤、腮和肠等8种组织中的都有表达，肝脏、脾脏、脑、头肾、肌肉、皮肤6种组织中表达量较高，心脏、鳃和肠3种组织中表达量较低，但是SCARA3基因在中的表达量最高。更优选，检测大黄鱼清道夫受体SCARA3基因的方法为：通过PCR扩增引物来扩增大黄鱼脾脏中的RNA。
上述大黄鱼清道夫受体SCARA3基因编码的大黄鱼清道夫受体蛋白，包括645个氨基酸，其氨基酸序列如SEQ ID NO:4所示。
上述大黄鱼清道夫受体蛋白的重组表达蛋白，将本发明还公开上述大黄鱼清道夫受体SCARA3基因连接于载体上，然后转入到宿主菌中，再用重组宿主进行表达制备大黄鱼清道夫受体蛋白。在本发明重组表达蛋白的制备过程中，载体在大肠杆菌内能够表达稳定，获得的重组蛋白对哈维弧菌的抑菌活性明显。
上述大黄鱼清道夫受体蛋白的重组表达蛋白在制备抑制哈维弧菌的生物制品中的用途。该生物制品为大黄鱼用饲料添加剂，能够提高大黄鱼对哈维弧菌的免疫力，这是因为大黄鱼清道夫受体蛋白具有显著的抑哈维弧菌活性，为大黄鱼抗哈维弧菌机理研究和抗哈维弧菌饲料添加剂的研制提供基因序列和方法。本发明的技术方法也可在其它鱼类上进行推广应用。
与现有技术相比，本发明的有益效果为：
本发明大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下，基因表达量上调，哈维弧菌感染后的识别较为敏感且反应迅速，最高表达是在感染后24h，感染后96h后仍有较高的表达，参与大黄鱼类的免疫过程；本发明大黄鱼清道夫受体SCARA3基因可用于筛选/检测大黄鱼抗哈维弧菌个体，尤其可以应用于大黄鱼亲鱼抗哈维弧菌力的检测和/或抗哈维弧菌良种的选育；本发明大黄鱼清道夫受体SCARA3基因编码的大黄鱼清道夫受体蛋白的重组表达蛋白具有显著的抑哈维弧菌活性，为大黄鱼抗哈维弧菌机理研究和抗哈维弧菌饲料添加剂的研制提供基因序列和方法。本发明的技术方法也可在其它鱼类上进行推广应用。
本发明采用了上述技术方案提供一种大黄鱼清道夫受体SCARA3基因及其用途，弥补了现有技术的不足，设计合理，操作方便。
附图说明
图1是本发明实施例1中大黄鱼清道夫受体SCARA3基因的ORF序列及推导的氨基酸序列；
图2是本发明实施例2中大黄鱼清道夫受体SCARA3基因在各组织中的表达；
图3是本发明实施例3中大黄鱼清道夫受体SCARA3基因在哈维弧菌感染后各组织中的表达；
图4是本发明实施例3中大黄鱼清道夫受体SCARA3基因在哈维弧菌感染后皮肤中的表达。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
下面，结合具体实施例对本发明实施方式作进一步说明。
实施例1：
大黄鱼清道夫受体SCARA3基因完整ORF通过如下方法获得：
S1：将收集得到的肝脏、脾脏、脑、心脏、头肾、肌肉、皮肤、腮和肠等8种组织样品分别进行总RNA提取；
(1)取30mg的组织样品于1.5mL的离心管中；
(2)在离心管中加入200μL Trizol Regent；
(3)用电研磨棒进行快速充分研磨后加入800μL Trizol Regent；
(4)用移液枪反复吹吸沉淀，使其重悬，室温放置5min；
(5)加入200μL氯仿，盖紧离心管，用手震荡剧烈混匀15s，室温放置10min；
(6)在4℃环境下，12000×g离心15min，使溶液分为上、中、下三层；
(7)小心吸取上层水相溶液至另一离心管中，加入500μL异丙醇，轻轻混匀；
(8)将混合后的溶液置于-20℃环境下30min；
(9)在4℃环境下，12000×g离心10min；
(10)弃去溶液，加入750μL 4℃预冷的75％乙醇进行洗涤；
(11)在4℃环境下，12000×g离心10min；
(12)重复步骤10和11；
(13)弃溶液后短暂离心，吸尽残余液体，置室温中数分钟，彻底晾干；
(14)向上述RNA沉淀中加入30μL RNase free dd H2O，轻微混匀使其溶解；
(15)将获得的总RNA以1.5％非变形琼脂糖凝胶电泳检测，并检测其浓度及A260/A280值，符合要求，可以进行后续实验。
S2：对S1提取的RNA进行反转录，获得相应的cDNA第一条链的合成；
反转反应体系为：5.0μL模板RNA、1.0μL Oligo(dT)，混匀离心后置于PCR仪上，扩增条件为70℃10min后0℃10min，取出短暂离心后，按下述体系加入：
混匀离心后置于PCR仪上40℃1h、70℃15min、0℃5min，即可获得cDNA第一链，同时反转3份，完成后置于-80℃超低温冰箱保存。
S3：以cDNA为模板进行PCR扩增，扩增大黄鱼清道夫受体SCARA3基因，纯化；
根据大黄鱼基因组数据库，通过Primer 5.0软件设计扩增完整清道夫受体家族基因ORF的引物(其上游引物的序列为SEQ ID NO:2，下游引物的序列为SEQ ID NO:3)，以cDNA为模板，扩增SCARA3基因，20μL扩增反应体系如下：
混匀离心后置于PCR仪上，扩增条件为：95℃预变性4min；94℃变性30s，55-65℃退火30s，72℃延伸2min，循环35次；最后72℃延伸10min。反应完成后PCR产物通过1.2％的琼脂糖凝胶进行电泳检验，以DL2000DNA marker作为标准的分子量标记，经PCR反应获得的DNA溶液用琼脂糖凝胶回收试剂盒进行纯化，具体步骤如下：
(1)柱平衡：向吸附柱CA2(吸附柱放入收集管中)中，加入500μL平衡液BL，12000rpm离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中；
(2)将单一的目的DNA条带从琼脂糖凝胶中切下(尽量切除多余部分)，放入干净的离心管中，称取重量；
(3)向胶中加入3倍体积(0.1g视为100μL)溶胶液PN，50℃水浴放置约10min，其间不断温和地上下翻转离心管，以确保胶块充分溶解；
(4)将上步所得溶液加入吸附柱CA2中，室温放置2min，12000rpm离心1min。超过800μL可分次加入，倒去收集管中的废液，将吸附柱CA2放入收集管中；
(5)加入600μL漂白液PW，12000rpm离心1min，倒掉废液，将吸附柱CA2放入收集管中；
(6)重复步骤5；
(7)将吸附柱放回管中，12000rpm离心2min，尽量除尽漂洗液，将吸附柱CA2置于室温放置数分钟，彻底晾干，以防止残留的漂洗液影响下一步的实验；
(8)将吸附柱放到一个干净的离心管中，向吸附膜中间位置悬空滴加30μL洗脱缓冲液EB，室温放置2min
(9)12000rpm离心2min，收集DNA溶液，置于-20℃低温冰箱保存；
(10)纯化完成后PCR产物通过1.2％的琼脂糖凝胶进行电泳检验，以DL2000 DNAmarker作为标准的分子量标记，应与纯化前产物的分子量相当。
S4：将S3纯化后产物进行T-A克隆，检测PCR产物，将含有合适片段大小的重组子进行DNA序列测序；
首先利用连接反应试剂盒TaKaRa pMD18-T Vector(TaKaRa，Japan)进行重组载体的构建，连接体系(总体积10μL)为：
Solution I 5.0μL
PCR产物 4.2μL
TaKaRa pMD18-T Vector 0.8μL
在冰上按体系中顺序逐一加入各物质，轻轻混匀后16℃连接过夜，连接完成后进行热激转化，具体步骤为：
(1)取感受态细胞DH5α，冰下融解5min，其间将100μL的枪头在冰箱里冰冻(-20℃)预冷，最后将100μL DH5α加入10μL连接产物中；
(2)打开水浴锅，42℃热激90s，然后迅速置于冰上静置3min；
(3)将PCR管中的液体(连接产物+DH5α)加入不含氨苄青霉素(Amp-)的LB液体培养基中，然后在37℃以180rpm振荡培养1-2h；
(4)在培养过程中，将40μL(20mg/ml)X-Gal和7μL(200mg/ml)IPTG均匀涂布在Amp+LB固体平板上，使其充分吸收；
(5)震荡培养结束后，将菌液以4000rpm离心5min，去除大部分上清液，吸取130μL菌液于步骤4准备好的Amp+LB固体平板上，均匀涂布，并标记；
(6)置于37℃培养过夜。(以上所有操作在超净工作台中进行)
将平板放于37℃恒温培养箱中培养过夜后即可出现菌落，培养后的平板再置于4℃下1h，用无菌牙签在每个平板中挑取白色单菌落(具体数量根据平板上实际菌落确定)，置于装有800μL Amp+的LB液体培养基的EP管中，编号，然后在37℃摇床以180rpm振荡培养约6h。以18T质粒载体通用引物M13R：5'-CGCCAGGGTTTTCCCAGTCACGAC-3'和M13F：5'-AGCGGATAACAATTTCA CACAGGA-3'作为PCR反应的引物进行菌液PCR扩增，10μL反应体系为：
混匀离心后置于PCR仪上。扩增条件为95℃预变性4min；94℃变性30s，56℃退火30s，72℃延伸2min，循环35次；最后72℃延伸7min。反应完成后以DL2000DNA marker作为标准的分子量标记，1.2％琼脂糖凝胶电泳检测PCR产物，将含有合适片段大小的重组子进行DNA序列测序，其完整ORF包含1938个碱基，核苷酸序列如SEQ ID NO:1或图1(*-终止信号；框-胶原样结构域；单线-TRAF2结合结构域，双线-酪氨酸结合结构域)所示。大黄鱼清道夫受体SCARA3基因和其他物种的SCARA3基因具有较高相似性，其理论分子量和等电点分别为70.94kDa和7.02，存在一个跨膜螺旋区(85-107aa)，两个α-螺旋区(147-219aa和326-354aa)以及一个胶原样结构域(519-584aa)，且存在3个Major tumor necrosis factorreceptor 2(TRAF2)因子，分别是T-G-E-E(58-61aa)、S-E-E-E(116-119aa)和A-D-E-E(267-270aa)，同时还存在2个酪氨酸活化区(Y-G-F-V，91-94aa和Y-Y-D-L，323-326aa)。大黄鱼清道夫受体SCARA3基因共有13个外显子，12个内含子。
实施例2：
大黄鱼清道夫受体SCARA3基因的组织特异性分布表达
首先取了健康大黄鱼(体长20-30cm，体重350-400g)的肝脏、脾脏、脑、心脏、头肾、肌肉、腮、肠和皮肤9个组织，根据清道夫受体SCARA3基因的测序结果，设计荧光定量PCR引物(如表1)，采用qRT-PCR法，以大黄鱼β-actin作为内参，分析SCARA3基因在各组织中的组织差异表达分布。20μL扩增反应体系如下：
混匀离心后置于荧光定量PCR仪上，采用两步法进行扩增：95℃预变性4min；95℃变性10s，60℃延伸45s，循环40次，最后72℃延伸10min。反应完成后PCR产物通过1.2％的琼脂糖凝胶进行电泳检验。结果如图2所示，我们发现虽然大黄鱼清道夫受体SCARA3基因在各个组织中广泛表达，但是又存在一定的组织特异性，在所选组织中，我们发现大黄鱼清道夫受体SCARA3基因在肝脏、脾脏、脑、头肾、肌肉、皮肤6种组织中表达量较高，心脏、鳃和肠3种组织中表达量较低，且SCARA3基因在脾脏中的表达量最高。
表1大黄鱼清道夫受体SCARA3基因荧光定量PCR引物序列
| Primer | Sequences |
| primer-F | 5'-CGATTGGCAGCATTACCATG-3' |
| primer-R | 5'-TCGGATAAGATATGGACGATG-3' |
| β-actin-F | 5'-TCGTCGGTCGTCCCAGGCATCAG-3' |
| β-actin-R | 5'-ATGGCGTGGGGCAGAGCGTAACC-3' |
实施例3：
大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下的表达
实验大黄鱼(体长20-30cm，体重350-400g)于25℃洁净海水中暂养1周，每天换新鲜海水。随后将大黄鱼随机分为2组，每组30条，其中实验组腹腔注射100μL PBS重悬的哈维弧菌菌液(pH 7.4，1×108CFU/mL)，对照组注射100μL PBS(pH 7.4)。收集注射后0h、2h、6h、12h、24h、48h、72h的肝脏、脾脏、脑、头肾、肌肉、皮肤6个组织，提取总RNA。用荧光定量PCR的方法检测大黄鱼清道夫受体SCARA3基因在感染哈维弧菌后0、6、12、24、48和72h的表达量，所用引物、试剂和方法均同实施例2介绍。采用2-ΔΔCt法计算大黄鱼清道夫受体SCARA3基因的相对表达量，结果如图3，我们发现，大黄鱼清道夫受体SCARA3基因对哈维弧菌感染后的识别较为敏感且反应迅速，在注射哈维氏弧菌后各组织的SCARA3基因表达水平均上调，其中在感染后24h，各组织的SCARA3基因表达量达最高，且SCARA3基因在脾脏中的表达量最高，同时在感染后72h后，各组织的SCARA3基因仍有较高的表达。以上结果表明：在哈维弧菌的刺激下，SCARA3基因参与大黄鱼类的免疫过程。
为了提高大黄鱼清道夫受体SCARA3基因对哈维弧菌感染后的识别敏感性，定量PCR检测用20μL扩增反应体系还包含0.1μL N-甲基甲酰胺。在注射哈维氏弧菌后，皮肤中的SCARA3基因表达量如图4所示，我们发现，大黄鱼皮肤中的清道夫受体SCARA3基因对哈维弧菌感染的识别敏感和反应速度均好于实施例2，且在感染后12h，皮肤中的SCARA3基因表达量达最高，同时在感染后72h和96h后的表达量高于实施例2。以上结果表明：定量PCR扩增反应体系中N-甲基甲酰胺的存在能够增强大黄鱼清道夫受体SCARA3基因对哈维弧菌感染后的识别敏感性。
实施例4：
一种大黄鱼清道夫受体SCARA3基因的检测方法，检测大黄鱼脾脏和/或肌肉和/或脑和/或肝脏中黄鱼清道夫受体SCARA3基因的表达量的方法，通过定量PCR扩增引物来扩增大黄鱼脾脏和/或肌肉和/或脑和/或肝脏中的RNA，所用引物、试剂和方法均同实施例2介绍。
实施例5：
大黄鱼清道夫受体SCARA3基因在筛选/检测大黄鱼抗哈维弧菌个体中的用途，按照常规定量PCR方法进行大黄鱼清道夫受体SCARA3基因表达水平分析，通过PCR扩增引物来扩增大黄鱼皮肤中的RNA，所用引物、试剂和方法均同实施例2介绍，比较不同大黄鱼成鱼或亲鱼皮肤的SCARA3基因表达水平，选取SCARA3基因表达水平高的成鱼进行养殖，或选取SCARA3基因表达水平高的亲鱼繁殖后代，筛选出抗病免疫能力强的大黄鱼个体，或生产出抗病免疫能力提高的鱼苗。本发明的方法首次将皮肤中SCARA3基因的表达水平作为抗病力评价的指标，在大黄鱼抗病力测试及抗病良种选育中具有重大应用价值，也可以在其它鱼类抗病育种上进行推广应用。
实施例6：
大黄鱼清道夫受体SCARA3基因编码的大黄鱼清道夫受体蛋白的重组表达蛋白的制备，包括如下步骤：
S1：将大黄鱼清道夫受体SCARA3基因连接于载体上：
(1)采用的载体是北京全式金公司的pEAZY-E1载体，根据大黄鱼清道夫受体SCARA3基因ORF的序列，设计了一对特异引物F:5’-ATTCACGAAAGATTTGCGACACCGA-3’；R:5’-TACTACGAGCTCTTCGCGAG-3’；
(2)以脾脏cDNA为模板，用常规的PCR方法对基因的ORF进行扩增，将扩增产物回收，经测序验证确定扩增序列无误后，连接到pEAZY-E1表达载体中；
(3)用热激法将连接好的质粒转到大肠杆菌TOP10菌株中，涂到含有100μg/mL氨苄青霉素的LB平板培养基上，37℃培养12h；
(4)挑取单克隆，加入1mL液体LB培养基，37℃震荡培养1小时后，以菌液为模板，用T7引物进行常规PCR扩增，以检测含有重组质粒pEAZY-E1-Pik的阳性克隆。所用引物为：T7-F:5’-TCGAACTATAAGCACTGTAG-3’和T7-R:5’-TGCGTATGCTTCGCTAGAG-3’。将阳性克隆扩大培养，提取质粒，获得SCARA3基因的重组大肠杆菌表达载体。
S2：重组蛋白的诱导表达与表达产物的分析：
(1)将载体转化到大肠杆菌BL21(DE3)plyS感受态中，挑取阳性单克隆到1ml含有终浓度50μg/mL氨苄青霉素的液体LB培养基中，37℃200rpm摇动培养8h到对数生长期；
(2)将菌液1:1000接种LB培养基37℃，200r/min过夜培养；
(3)再按照1:100比例将菌液加入LB培养基扩培37℃200r/min培养至OD600为0.6～0.8，加入终浓度为1mM的IPTG，20℃诱导过夜；
(4)取约3ml菌液离心10min收集菌体沉淀，用200μL预冷的PBS洗涤，12000rpm，1min离心后，用30μL PBs悬浮，并加入10μL 4×蛋白上样缓冲液，煮沸5min，冷却后用SDS-PAGE垂直电泳检测，电泳电压设置为：180V，1h。检测得到相应的目的条带，则进行大规模的诱导，诱导好的菌液同样进行离心，PBS冲洗。
S3：重组蛋白分离、纯化和验证
在收集的菌体沉淀中加入20mL裂解液(25mmol/L Tris，5mmol/L EDTA 50mmol/LNaCl；pH7.5)，同时加入0.3mg/mL的溶菌酶和终浓度为1mmol/L的PMSF(phenyl methylsulfonyl fluoride)，充分混合均匀，冰上放置2h，超声破碎30min(破碎0.5s，间隔2s)。破碎结束后，8000rpm，低温离心30min，将上清和沉淀分离分别置于不同的收集管中，分别对上清液以及沉淀进行SDS-PAGE蛋白电泳，检测到表达蛋白是以包涵体的形式存在。再用20mL溶液A(50mmol/L Tris-HCl，2mol/L尿素，0.5mmol/L EDTA，100mmol/L NaCl，1％Triton X-100；pH 8.0)洗涤两次，每次洗涤后在12000rpm，4℃离心30min，弃上清。再用溶液B(50mmol/L Tris-HCl，2mol/L尿素，0.5mmol/L EDTA，100mmol/L NaCl；pH 8.0)洗涤两次，并同样离心。洗涤结束后的沉淀用预冷的溶解液(100mmol/L Tris、8mmol/L尿素、10mmol/L咪唑、500mmol/L NaCl；pH 7.4)4℃冰箱中磁力搅拌溶解2h至溶液透明，12000rpm，4℃离心30min。离心得到的上清用0.22μm滤膜过滤除菌后，使用HisTrapFFcrude 1ml纯化柱进行纯化，得重组蛋白。重组蛋白经western-blotting验证，结果显示纯化蛋白条带单，符合预测大小(70.8KD)。
上述实施例中的常规技术为本领域技术人员所知晓的现有技术，故在此不再详细赘述。
以上实施方式仅用于说明本发明，而并非对本发明的限制，本领域的普通技术人员，在不脱离本发明的精神和范围的情况下，还可以做出各种变化和变型。因此，所有等同的技术方案也属于本发明的范畴，本发明的专利保护范围应由权利要求限定。
序列表
<110> 浙江海洋大学
<120> 大黄鱼清道夫受体SCARA3基因及其用途
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1938
<212> DNA
<213> 大黄鱼(Larimichthys crocea)
<400> 1
atggcgacgc aggctgtaaa gaaaacagag acctacgcaa gcttctggtg gctccaagtg 60
gccaaaaaag ttactgcaga tttacatctt tttctccatt ttggcagtat tcatgcacat 120
gcagataact atggtggata tgagaaccag ctttttaaag aggaggattt cactggagaa 180
gaggaaatgc cttcatttag aggtcgtagc agaggaggct gcatgaggtg tcagcagagc 240
cactctctcc agctggctgt caaagtcctc tatggatttg ttgcttttct catcattaca 300
gtggcagtgc tggcatcgct tgtgttcagg aaggtggaca gcttgtctga ggaggagtcc 360
gtctaccaca agaagatctc cagagttcag caaggaatag atgatctgag ctccagctct 420
aactgttcag gctgtctgga tgtggctctg tacagccagc agatcagcag gctgaagaga 480
gagtttgaag acatccagag aatgattctg ggacaggagc aggtcctgga ccaggcctcc 540
cagacccaga acaccctaaa ggccaccagc aaccggctga cacgcgacat gcaaaaccac 600
ctcatgtcta tcaaaattct caaccagtca ctggaaagat acctggatcg ggtggaaggc 660
tggaaggacg tgatcgagga aactgtaggg aaaatgaaga cactggcgga tgatcagtat 720
gacgtcaaag ctacagcaca gcaagttaac atgacggtgg cactcagtac aatgtggata 780
gacgccctgc agagaaaagc agacgaggag actctggtcc tccagaagtt gaccaccgac 840
tggcagaact acagtcgagt tctgaactcc attaaatcca acacaagcag caccacgcaa 900
agcatgcgtt tcctgcaaaa caacattgca gcagatcacc agagaatcgc catgtccaat 960
gaggtgtact acgacctcac ccagcaggtg atgaacttgc agatgcagct tgacaacgtg 1020
acgtctttca tggatgaaca cgaggagaac atgcatgacc ttcactacca ttccaggtac 1080
tatgagaacc ggactggtga gcgtttttct gcgttggatg gtcgtctgaa ctccatctcg 1140
atggagattg atacgatctc ctccagcatc aatgccactg tcaaccacgt ccagagcatg 1200
tacaagtaca tcaacatcga gagctcatcc tgccagagtc gcatgggcag acacacagaa 1260
gatctacaga acctgaacaa cacagttttg ttacttctcc atttggcgga cacactgaga 1320
cagcaaaaca tgttgctgaa tctaagactg gatgtggatg tcagaaacct gtccatggtg 1380
atggaggaga tgaagcttgt ggatgttcac catacacagc tcataaagaa cttcactata 1440
gtcaaaggta ttcctggacc accagggccc aaagggaacc gtggtgagac tggctcaaaa 1500
ggacccatgg gactaactgg gagcaaaggt gacagaggcc cgacaggaag ccgtggatta 1560
actggagaga agggttcttt agggcccata ggagcaacag gtgcaccagg gcccagtggc 1620
aacaaagggt caattggact caaaggggct aaagggtctc ccggttttcc aggaacacgt 1680
ggcgagaagg gccagaaagg tgacatgggc ctctctggtg ctgatggcat agaaggactc 1740
acagggcctc cagggatcca gggtcaagca ggacaaccag gcgttattgg gccagtagga 1800
ccaaggggaa aaccagggcc agcagggcca cctggaccac caggaacccc tggatctcca 1860
ggcttgccat acccccatga ccgaatcaac acccaagagc ggaccgtgac gcctgctact 1920
gggtccaaaa gacagtag 1938
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agagtgactc gagctctagc g 21
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtattgactt atggagccag catt 24
<210> 4
<211> 645
<212> PRT
<213> 大黄鱼(Larimichthys crocea)
<400> 4
Met Ala Thr Gln Ala Val Lys Lys Thr Glu Thr Tyr Ala Ser Phe Trp
1 5 10 15
Trp Leu Gln Val Ala Lys Lys Val Thr Ala Asp Leu His Leu Phe Leu
20 25 30
His Phe Gly Ser Ile His Ala His Ala Asp Asn Tyr Gly Gly Tyr Glu
35 40 45
Asn Gln Leu Phe Lys Glu Glu Asp Phe Thr Gly Glu Glu Glu Met Pro
50 55 60
Ser Phe Arg Gly Arg Ser Arg Gly Gly Cys Met Arg Cys Gln Gln Ser
65 70 75 80
His Ser Leu Gln Leu Ala Val Lys Val Leu Tyr Gly Phe Val Ala Phe
85 90 95
Leu Ile Ile Thr Val Ala Val Leu Ala Ser Leu Val Phe Arg Lys Val
100 105 110
Asp Ser Leu Ser Glu Glu Glu Ser Val Tyr His Lys Lys Ile Ser Arg
115 120 125
Val Gln Gln Gly Ile Asp Asp Leu Ser Ser Ser Ser Asn Cys Ser Gly
130 135 140
Cys Leu Asp Val Ala Leu Tyr Ser Gln Gln Ile Ser Arg Leu Lys Arg
145 150 155 160
Glu Phe Glu Asp Ile Gln Arg Met Ile Leu Gly Gln Glu Gln Val Leu
165 170 175
Asp Gln Ala Ser Gln Thr Gln Asn Thr Leu Lys Ala Thr Ser Asn Arg
180 185 190
Leu Thr Arg Asp Met Gln Asn His Leu Met Ser Ile Lys Ile Leu Asn
195 200 205
Gln Ser Leu Glu Arg Tyr Leu Asp Arg Val Glu Gly Trp Lys Asp Val
210 215 220
Ile Glu Glu Thr Val Gly Lys Met Lys Thr Leu Ala Asp Asp Gln Tyr
225 230 235 240
Asp Val Lys Ala Thr Ala Gln Gln Val Asn Met Thr Val Ala Leu Ser
245 250 255
Thr Met Trp Ile Asp Ala Leu Gln Arg Lys Ala Asp Glu Glu Thr Leu
260 265 270
Val Leu Gln Lys Leu Thr Thr Asp Trp Gln Asn Tyr Ser Arg Val Leu
275 280 285
Asn Ser Ile Lys Ser Asn Thr Ser Ser Thr Thr Gln Ser Met Arg Phe
290 295 300
Leu Gln Asn Asn Ile Ala Ala Asp His Gln Arg Ile Ala Met Ser Asn
305 310 315 320
Glu Val Tyr Tyr Asp Leu Thr Gln Gln Val Met Asn Leu Gln Met Gln
325 330 335
Leu Asp Asn Val Thr Ser Phe Met Asp Glu His Glu Glu Asn Met His
340 345 350
Asp Leu His Tyr His Ser Arg Tyr Tyr Glu Asn Arg Thr Gly Glu Arg
355 360 365
Phe Ser Ala Leu Asp Gly Arg Leu Asn Ser Ile Ser Met Glu Ile Asp
370 375 380
Thr Ile Ser Ser Ser Ile Asn Ala Thr Val Asn His Val Gln Ser Met
385 390 395 400
Tyr Lys Tyr Ile Asn Ile Glu Ser Ser Ser Cys Gln Ser Arg Met Gly
405 410 415
Arg His Thr Glu Asp Leu Gln Asn Leu Asn Asn Thr Val Leu Leu Leu
420 425 430
Leu His Leu Ala Asp Thr Leu Arg Gln Gln Asn Met Leu Leu Asn Leu
435 440 445
Arg Leu Asp Val Asp Val Arg Asn Leu Ser Met Val Met Glu Glu Met
450 455 460
Lys Leu Val Asp Val His His Thr Gln Leu Ile Lys Asn Phe Thr Ile
465 470 475 480
Val Lys Gly Ile Pro Gly Pro Pro Gly Pro Lys Gly Asn Arg Gly Glu
485 490 495
Thr Gly Ser Lys Gly Pro Met Gly Leu Thr Gly Ser Lys Gly Asp Arg
500 505 510
Gly Pro Thr Gly Ser Arg Gly Leu Thr Gly Glu Lys Gly Ser Leu Gly
515 520 525
Pro Ile Gly Ala Thr Gly Ala Pro Gly Pro Ser Gly Asn Lys Gly Ser
530 535 540
Ile Gly Leu Lys Gly Ala Lys Gly Ser Pro Gly Phe Pro Gly Thr Arg
545 550 555 560
Gly Glu Lys Gly Gln Lys Gly Asp Met Gly Leu Ser Gly Ala Asp Gly
565 570 575
Ile Glu Gly Leu Thr Gly Pro Pro Gly Ile Gln Gly Gln Ala Gly Gln
580 585 590
Pro Gly Val Ile Gly Pro Val Gly Pro Arg Gly Lys Pro Gly Pro Ala
595 600 605
Gly Pro Pro Gly Pro Pro Gly Thr Pro Gly Ser Pro Gly Leu Pro Tyr
610 615 620
Pro His Asp Arg Ile Asn Thr Gln Glu Arg Thr Val Thr Pro Ala Thr
625 630 635 640
Gly Ser Lys Arg Gln
645
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agcggataac aatttcacac agga 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agcggataac aatttcacac agga 24
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgattggcag cattaccatg 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcggataaga tatggacgat g 21
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcgtcggtcg tcccaggcat cag 23
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggcgtggg gcagagcgta acc 23
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
attcacgaaa gatttgcgac accga 25
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tactacgagc tcttcgcgag 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tcgaactata agcactgtag 20
<210> 14
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tgcgtatgct tcgctagag 19
Claims (10)
1.大黄鱼清道夫受体SCARA3基因，其特征在于：其完整ORF包含1938个碱基，核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的大黄鱼清道夫受体SCARA3基因，其特征在于：所述大黄鱼清道夫受体SCARA3基因完整ORF通过如下方法获得：
S1：将收集得到的各类样品进行总RNA提取；
S2：对S1提取的RNA进行反转录，获得相应的cDNA第一条链的合成；
S3：以cDNA为模板进行PCR扩增，扩增大黄鱼清道夫受体SCARA3基因，纯化；
S4：将S3纯化后产物进行T-A克隆，检测PCR产物，将含有合适片段大小的重组子进行DNA序列测序。
3.根据权利要求2所述的大黄鱼清道夫受体SCARA3基因，其特征在于：所述PCR扩增用引物，其上游引物的序列为SEQ ID NO:2，下游引物的序列为SEQ ID NO:3。
4.根据权利要求1或2或3所述的大黄鱼清道夫受体SCARA3基因，其特征在于：所述大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下，基因表达量明显升高。
5.大黄鱼清道夫受体SCARA3基因在筛选/检测大黄鱼抗哈维弧菌个体中的用途，其特征在于：所述大黄鱼清道夫受体SCARA3基因在哈维弧菌的感染下，基因表达量上调。
6.一种大黄鱼清道夫受体SCARA3基因的检测方法，其特征在于：检测大黄鱼脾脏和/或肌肉和/或皮肤和/或脑和/或肝脏中权利要求1-4任一项所述基因的表达量的方法。
7.根据权利要求6所述的一种大黄鱼清道夫受体SCARA3基因的检测方法，其特征在于：所述检测方法通过PCR扩增引物来扩增大黄鱼脾脏和/或肌肉和/或皮肤和/或脑和/或肝脏中的RNA。
8.权利要求1-4任一项所述的大黄鱼清道夫受体SCARA3基因编码的大黄鱼清道夫受体蛋白，其特征在于：包括645个氨基酸，其氨基酸序列如SEQ ID NO:4所示。
9.权利要求8所述大黄鱼清道夫受体蛋白的重组表达蛋白，其特征在于：将权利要求1-5任一项所述大黄鱼清道夫受体SCARA3基因连接于载体上，然后转入到宿主菌中，再用重组宿主进行表达制备大黄鱼清道夫受体蛋白。
10.权利要求9所述大黄鱼清道夫受体蛋白的重组表达蛋白在制备抑制哈维弧菌的生物制品中的用途。
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| CN112391389A (zh) * | 2020-11-19 | 2021-02-23 | 山西大学 | 飞蝗C型清道夫受体基因及其靶向dsRNA与应用 |
| CN114990158A (zh) * | 2022-05-19 | 2022-09-02 | 北京大学 | Scara3基因敲除仓鼠模型的构建方法 |
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| CN112391389A (zh) * | 2020-11-19 | 2021-02-23 | 山西大学 | 飞蝗C型清道夫受体基因及其靶向dsRNA与应用 |
| CN112391389B (zh) * | 2020-11-19 | 2022-09-23 | 山西大学 | 飞蝗C型清道夫受体基因及其靶向dsRNA与应用 |
| CN114990158A (zh) * | 2022-05-19 | 2022-09-02 | 北京大学 | Scara3基因敲除仓鼠模型的构建方法 |
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