CN116178521B - 一种鱼源肽聚糖识别蛋白突变体、制备方法及其应用 - Google Patents
一种鱼源肽聚糖识别蛋白突变体、制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种鱼源肽聚糖识别蛋白突变体、制备方法及其应用,属于分子生物学技术领域,所述突变体的序列如SEQ ID NO.1所示,所述突变体的核苷酸序列如SEQ ID NO.2所示。本发明还提供所述突变体的制备方法,所述突变体具有广谱的抗菌活性,对革兰氏阳性菌和革兰氏阴性菌都具有较强的抑制作用。和野生型LcPGRP5蛋白相比,突变体蛋白抑制金黄色葡萄球菌的活性更强。且增强巨噬细胞的吞噬能力。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种新型的鱼源肽聚糖识别蛋白突变体的制备方法及其应用。
背景技术
硬骨鱼作为低等的脊椎动物具有功能较为完善的先天性免疫系统,能够通过模式识别受体(PRR)识别细菌和病毒的入侵和感染,介导免疫细胞发挥免疫调节功能。研究表明,肽聚糖识别蛋白(PGRP),一个具有多种生理功能的肽聚糖识别蛋白家族,被认为在鱼类固有免疫系统对抗微生物感染中起主导作用。
PGRPs是在进化上从昆虫到哺乳动物都比较保守的PRR,具有一个160氨基酸的PGRP结构域。哺乳动物中发现四种基因组成的PGRP家族,昆虫则有更多(例如,果蝇中编码19种蛋白质的13个基因),据报道,鱼类中有三种类型的PGRP基因,分别被命名为PGRP2、PGRP5(PGRP-SC)和PGRP6。
在昆虫和哺乳动物的研究表明,PGRP通过直接作用(杀菌、抗菌活性)和间接作用(诱导抗菌肽以及调节炎症和免疫反应)控制哺乳动物和昆虫机体的共生菌水平,并抵御其他细菌的感染。同样,在鱼类中的研究表明,PGRP在病原体识别、微生物凝集、诱导凋亡和促进吞噬等免疫调节功能中发挥着重要作用。此外,鱼类PGRP还是一种酰胺酶,可以水解肽聚糖分子中N-乙酰壁酸和丙氨酸之间的酰胺键,导致肽聚糖分子失去活性,达到杀灭细菌的目的。越来越多的证据表明PGRP还能够与微生物表面的肽聚糖发生相互作用,激活下游级联信号转导,激活炎症介质的产生,是清除病原体的关键免疫效应分子。
近几十年来,哈维氏弧菌、溶藻弧菌、丝状诺卡氏菌和假单胞菌等病原菌引起的传染病的暴发,造成了严重的经济损失,对鱼类养殖的可持续发展提出了巨大挑战。如何突破这一“瓶颈”,寻找调控鱼类免疫力的关键靶点,开发提高养殖鱼类免疫和健康的精准调控策略,对保障水产养殖业的健康、可持续发展具有重要的意义。已有的研究表明,LPS或灭活细菌疫苗能够显著提高大黄鱼PGRP5的表达,表明PGRP5在细菌所诱导的免疫过程中发挥重要的作用,这为制定通过PGRPs的调控策略以促进水产养殖实践中的健康管理提供了重要的理论依据。并且,目前已经有通过基因工程方法生产重组鱼类肽聚糖识别蛋白的报道,但仍存在活性发挥的机制不清晰、生物活性不高以及应用场景受限等问题。
发明内容
本发明的目的是提供一种鱼类肽聚糖识别蛋白突变体、制备方法及应用,对其活性功能进行分析,并应用于营养免疫领域。
本发明是通过以下技术方案实现的:
一种鱼类肽聚糖识别蛋白突变体,所述突变体的氨基酸序列如SEQ ID NO.1所示。进一步,所述鱼类肽聚糖识别蛋白突变体的核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.1
MDQKVNIVS RVQWGAAAPR KKETLKDCAQ RVVIHHTALP KCTGMKECVD RLVSIQRAHMTERRFDDIGY NFLVGGDGTV YEGRGWGVVG AHTKGHNHDS LGIAFMGNYN SDAPSTEALS AVKQLLQSGVSQGFLQPEFV LFGARDLGST QCPGDKLYAA LPQLRGTT;
SEQ ID NO.2
atggatcagaaagttaacatcgttagtcgcgttcagtggggcgcagcagcaccgcgtaaaaaagaaaccctgaaagattgcgcacagcgcgttgttattcatcataccgcactgccgaaatgtaccggcatgaaagaatgtgttgatcgtctggtgagcattcagcgcgcccacatgaccgaacgtcgttttgatgatattggttataatttcctggtgggcggtgatggtaccgtgtatgaaggtcgtggttggggtgttgttggcgcacataccaaaggccataatcatgatagtctgggcattgcctttatgggcaattataatagtgatgccccgagtaccgaagccctgagcgcagttaaacagctgctgcagagtggcgtgagtcagggttttctgcagccggaatttgtgctgtttggtgcacgcgatctgggtagtacccagtgtccgggtgataaactgtatgcagccctgccgcagctgcgtggcaccacataa。
本发明还提供鱼类肽聚糖识别蛋白突变体的制备方法,以所述突变体基因为模板进行PCR反应扩增目的片段,得到带有接头的目标基因,采用同源重组方式将扩增后的PCR产物连接到原核表达系统载体pET-PDE1,将重组突变体质粒导入到大肠杆菌表达系统中,筛选并获得高效表达菌株,接种于添加抗生素的培养基中,加入IPTG诱导重组融合蛋白在细菌中表达,继续培养离心收集菌体沉淀,裂解菌体后收集包涵体,经过洗涤、变性溶解、透析复性后,采用亲和层析法对上清液进行纯化带有组氨酸标签的重组蛋白。
本发明还提供所述鱼类肽聚糖识别蛋白突变体在制备抗菌剂中的应用。
本发明还提供所述肽聚糖识别蛋白突变体在制备水产动物免疫增强剂或饲料添加剂中的应用。
本发明与现有技术相比的有益效果:
A本发明所制备的新型肽聚糖识别蛋白突变体是针对天然肽聚糖识别蛋白的氨基酸进行定点突变,从而改善了其生物活性。
B本发明中的新型肽聚糖识别蛋白突变体基因经过密码子优化,从而有助于密码子在大肠杆菌细胞中的识别和表达,进而提高原核表达重组蛋白的效率。
C本发明中将肽聚糖识别蛋白突变体重组质粒导入到大肠杆菌表达系统中,筛选并获得高效表达菌株。经过诱导、分离、纯化即可获得高活性的重组突变体蛋白,生产流程经过优化具备大规模生产的潜力。
D本发明中制备的肽聚糖识别蛋白突变体具有较强的酰胺酶活性,能够降解L型和D型肽聚糖,尤其对来自革兰氏阳性菌的L-型肽聚糖的降解能力更强。
E本发明中制备的肽聚糖识别蛋白突变体具有广谱的抗菌活性,对革兰氏阳性菌和革兰氏阴性菌都具有较强的抑制作用。
F本发明中制备的肽聚糖识别蛋白突变体具有增强的抗菌活性,和野生型LcPGRP5蛋白相比,突变体蛋白抑制金黄色葡萄球菌的活性更强。
G本发明中制备的肽聚糖识别蛋白突变体能够增强巨噬细胞的吞噬活性。添加重组突变体蛋白的巨噬细胞对金黄色葡萄球菌的吞噬率(70.7%)显著高于添加野生型rLcPGRP5的处理组(57.2%)和对照组(48%)。
附图说明
图1为纯化的突变体蛋白的SDS-PAGE分析图和Western blot鉴定分析图。
图2为本发明制备的突变体蛋白降解肽聚糖的酰胺酶活性图。
图3为本发明制备的突变体蛋白抑菌活性图。
图4为本发明制备的突变体蛋白增强免疫细胞吞噬能力图。
具体实施方式
本发明一种新型的鱼源肽聚糖识别蛋白突变体的制备方法及其应用。首先克隆得到天然的LcPGRP5的基因,并对其进行原核表达的密码子优化。对特定氨基酸的碱基序列进行突变,全基因合成突变体基因。采用采用定向拓扑异构酶克隆技术将突变体连接到原核表达系统载体pET-PDE1。将该重组突变体质粒导入到大肠杆菌表达系统中,筛选并获得高效表达菌株。进一步诱导重组融合蛋白在细菌中表达,经过裂解和纯化后获得突变的肽聚糖识别蛋白。
上述方法的具体步骤如下:
1、首先参考GenBank中预测的LcPGRP5序列(GenBank:MW468048.1)设计引物(所述特异性引物分别为:F:ATGGACCAAAAAGTGAACATTG;R:TTATGTGGTACCCCTCAGTTGTGG),以大黄鱼CDNA为模板,利用TAKARA公司的PRIMERSTARMAX酶按照98℃反应10s,55℃反应15s,72℃反应1MIN进行,共计35个循环的PCR扩增,PCR产物经1.2%琼脂糖凝胶电泳分离,回收目的片段大小的凝胶块,按照胶回收试剂盒的步骤进行胶回收。将回收的PCR产物连接到PEASY-BLUNT载体上(全式金生物技术有限公司,中国),转化TRANS-T1感受态细胞过夜培养后挑选阳性单克隆菌落测序验证。克隆得到野生型LCPGRP5基因的CDS区域。
2、对克隆得到的基因进行原核表达的密码子优化并对目标氨基酸密码子进行突变,全基因合成得到突变体基因。设计带有接头的正向和反应引物(F:CACCATGGACCAAAAAGTGAACATTG;R:
TTATGTGGTACCCCTCAGTTGTGG),以经过密码子优化的突变体基因为模板进行PCR反应扩增目的片段,得到带有接头的目标基因扩增产物,采用定向拓扑异构酶克隆技术将扩增纯化后的PCR产物定向克隆到表达载体pET-PDE1。反应体系(10μL)如下:0.5-8μL纯化后的PCR产物,载体1μL,1μL 10×Enhancer,其余用DEPC水补齐到10μL,室温(20℃-30℃)连接5分钟。构建得到表达产物带有6个组氨酸的原核表达载体。
3、采用氯化钙法转化质粒并筛选得到阳性转化子。将5-10μl连接反应产物加入100μl感受态细胞DH5α内,于冰上放置30min。取出后置于42℃水浴中热激50s,然后立即将反应物移回至冰上放置1min。加入300-500μl LB液体培养基(不含抗生素),于37℃空气浴恒温摇床上220rpm速度下振荡培养1h。取振荡培养的菌液200μl涂布于含有50μg/ml抗生素平板上,用涂布棒涂布均匀直至菌液完全被平板吸收,然后置于培养箱内37℃倒置培养过夜。设计上下游引物或质粒的通用引物对转化菌进行菌落PCR,PCR反应模板以灭菌牙签或灭菌10μl枪头挑取单菌落作为模板,琼脂糖凝胶电泳检测PCR产物。PCR检测阳性克隆进一步送生工测序。
4、将重组表达载体转化得到高表达大肠杆菌菌株,并进行重组蛋白诱导表达、鉴定与纯化。将重组质粒导入到大肠杆菌BL21(全式金,中国),挑取单克隆,于添加卡那霉素(50ug/mL)的LB培养基中,220rpm,37℃培养至OD600约为0.6时,加入终浓度为0.1mM的IPTG(索莱宝,中国),继续培养4-6h。随后,于4℃,6000rpm,离心10min,收集菌体沉淀。加入上样缓冲液后沸水浴中裂解10min后离心,取上清,于15%的SDS-PAGE电泳分析,考马斯亮蓝染色的方法初步验证重组蛋白的表达。
采用金属螯合层析方法纯化重组蛋白,利用组氨酸侧链上的咪唑基与金属离子(Ni2+)等螯合的特性,使基因重组技术制备的His-Tag融合重组蛋白得到纯化(金斯瑞,中国)。从镍柱洗脱下来的蛋白通过配有Ultracel-30滤膜的Amicon Ultra-15离心式过滤器(Millipore,美国)进行蛋白浓缩,之后透析(20mM Tris-HCl,0.15M氯化钠,pH 8.0)去掉多余的盐分和咪唑。透析后的蛋白经过冷冻干燥后得到纯化蛋白,并通过SDS-PAGE电泳和Westernblot分析以及考马斯亮蓝染色结合质谱分析的方法验证重组蛋白表达正确,如图1所示,通过Bradford法进行测定蛋白浓度(索莱宝,中国)。
5、肽聚糖识别蛋白突变体酰胺酶活性的测定。以紫外分光光度法检测检测野生型肽聚糖识别蛋白以及其突变体蛋白的酰胺酶活性。将不溶性的肽聚糖(1mg/mL,来自金黄色葡萄球菌)分别与以下溶液进行孵育:野生型肽聚糖识别蛋白(0.5mg/mL)+Tris-ZnCl2溶液(20mM Tris-HCl,pH=7.2,150mM NaCl,10mM ZnCl2)、突变型肽聚糖识别蛋白(0.5mg/mL)+Tris-ZnCl2溶液,室温下震荡孵育。120min内每隔5min测定并记录OD540值。和野生型LcPGRP5相比,突变体蛋白的酰胺酶活性更强,如图2所示,可显著降低PGN溶液的OD540值。
6、重组蛋白的抑菌实验。采用牛津杯打孔法方法检测重组蛋白的抑菌活性。37℃培养条件下,在LB液体培养基中大肠杆菌和金黄色葡萄球菌培养至对数生长期时,取0.2mL的菌体加入到固体培养基倒平板。铺设平板后利用牛津杯在平皿中打2-4个孔,分别在孔中加入50μg重组蛋白,培养12h后测量抑菌圈大小。野生型和突变体肽聚糖识别蛋白都具有抑制金黄色葡萄球菌的活性,且突变体重组蛋白的抑菌活性显著高于野生型重组蛋白,如图3所示。
7、LcPGRP5突变体蛋白对鱼类巨噬细胞吞噬能力的影响。利用重组蛋白孵育大黄鱼巨噬细胞,检测其对巨噬细胞吞噬荧光标记的细菌能力的影响。制备FITC标记的细菌。D-氨基酸缀合荧光素(FITC-d-Lys,Bioluminor,上海)与对数期的大肠杆菌和金黄色葡萄球菌在培养基中孵育12h,探针被菌体吸收后,以共价键的形式连接在细菌的细胞壁上,离心去上清,0.5%的福尔马林过夜杀灭细菌,PBS离心洗涤三遍后,加入少量PBS重悬细菌待用。利用纯化的重组蛋白(0.1-1mg/L)孵育大黄鱼巨噬细胞,孵育12-24h后加入已经灭活的大肠杆菌或者金黄色葡萄球菌继续孵育6h,加入预冷的培养基终止细胞吞噬后,胰蛋白酶消化贴壁的巨噬细胞到流式分析管中,置于冰上,用流式细胞仪(BD,美国)检测和记录荧光(激发光488nm)。吞噬能力按照每10000个细胞中发生吞噬的细胞比例。突变体蛋白显著促进了巨噬细胞对金黄色葡萄球菌的吞噬能力。孵育重组突变体蛋白后的巨噬细胞对金黄色葡萄球菌的吞噬率(70.7%)显著高于孵育野生型rLcPGRP5的处理组(57.2%)和对照组(48%),如图4所示。
Claims (4)
1.一种鱼类肽聚糖识别蛋白突变体,其特征在于,所述突变体的氨基酸序列如SEQ IDNO.1所示,所述突变体的编码基因的核苷酸序列如SEQ ID NO.2所示。
2.权利要求1所述鱼类肽聚糖识别蛋白突变体的制备方法,其特征在于,以权利要求1所述突变体的基因为模板进行PCR反应扩增目的片段,得到带有接头的目标基因,采用同源重组方式将扩增后的PCR产物连接到原核表达系统载体pET-PDE1,将重组突变体质粒导入到大肠杆菌表达系统中,筛选并获得高效表达菌株,接种于添加抗生素的培养基中,加入IPTG诱导重组融合蛋白在细菌中表达,继续培养离心收集菌体沉淀,裂解菌体后收集包涵体,经过洗涤、变性溶解、透析复性后,采用亲和层析法对上清液进行纯化带有组氨酸标签的重组蛋白。
3.权利要求1所述鱼类肽聚糖识别蛋白突变体在制备抗菌剂中的应用。
4.权利要求1所述所述肽聚糖识别蛋白突变体在制备水产动物免疫增强剂或饲料添加剂中的应用。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1982456A (zh) * | 2006-04-29 | 2007-06-20 | 中山大学 | 中国文昌鱼肽聚糖识别蛋白B.b.PGRP及其制备方法和应用 |
CN101619102A (zh) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | 一种具杀菌活性的栉孔扇贝肽聚糖识别蛋白的制备及应用 |
CN103131686A (zh) * | 2011-11-29 | 2013-06-05 | 中国科学院海洋研究所 | 一种二型肽聚糖识别蛋白及其制备和应用 |
CN104531712A (zh) * | 2014-12-07 | 2015-04-22 | 浙江大学 | 具有杀菌活性的烟粉虱肽聚糖识别蛋白的制备及应用 |
CN106749619A (zh) * | 2017-02-13 | 2017-05-31 | 盐城工学院 | 一种短型肽聚糖识别蛋白及其制备方法、分离的核酸以及应用和抗菌药物 |
CN113583102A (zh) * | 2021-08-28 | 2021-11-02 | 青岛农业大学 | 一种牙鲆肽聚糖识别蛋白的重组蛋白及其制备方法和应用 |
-
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Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1982456A (zh) * | 2006-04-29 | 2007-06-20 | 中山大学 | 中国文昌鱼肽聚糖识别蛋白B.b.PGRP及其制备方法和应用 |
CN101619102A (zh) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | 一种具杀菌活性的栉孔扇贝肽聚糖识别蛋白的制备及应用 |
CN103131686A (zh) * | 2011-11-29 | 2013-06-05 | 中国科学院海洋研究所 | 一种二型肽聚糖识别蛋白及其制备和应用 |
CN104531712A (zh) * | 2014-12-07 | 2015-04-22 | 浙江大学 | 具有杀菌活性的烟粉虱肽聚糖识别蛋白的制备及应用 |
CN106749619A (zh) * | 2017-02-13 | 2017-05-31 | 盐城工学院 | 一种短型肽聚糖识别蛋白及其制备方法、分离的核酸以及应用和抗菌药物 |
CN113583102A (zh) * | 2021-08-28 | 2021-11-02 | 青岛农业大学 | 一种牙鲆肽聚糖识别蛋白的重组蛋白及其制备方法和应用 |
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