CN112300259A - 一种自噬蛋白beclin-1及其编码基因和应用 - Google Patents

一种自噬蛋白beclin-1及其编码基因和应用 Download PDF

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CN112300259A
CN112300259A CN202011231537.0A CN202011231537A CN112300259A CN 112300259 A CN112300259 A CN 112300259A CN 202011231537 A CN202011231537 A CN 202011231537A CN 112300259 A CN112300259 A CN 112300259A
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李军
周银银
张跃环
喻子牛
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South China Sea Institute of Oceanology of CAS
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Abstract

本发明公开了一种自噬蛋白beclin‑1及其编码基因和应用。自噬蛋白beclin‑1，氨基酸序列如SEQ ID NO.2所示。本发明首次从香港牡蛎中鉴定获得Beclin‑1基因，干扰Beclin‑1的表达可以抑制自噬体的形成，抑制NF‑κB信号通路，而且可以快速响应溶藻弧菌、溶血葡萄球菌的感染。本发明中的Beclin‑1在香港牡蛎免疫防御中发挥重要的作用，为牡蛎及其它海水养殖贝类的病害防治、抗病育种提供应用指导。

Description

一种自噬蛋白beclin-1及其编码基因和应用
技术领域
本发明涉及应用海洋生物技术领域，更具体涉及一种自噬蛋白Beclin-1及其编码基因和应用。
背景技术
香港牡蛎(Crassostrea hongkongensis)属于属软体动物门(Mollusca)、双壳纲(Bivalvia)、珍珠贝目(Pterioidae)，牡蛎科(Ostridae)、巨蛎属(Crassostrea)，是我国一种重要的经济养殖贝类。主要分布于中国广西、广东、福建和海南沿海河口附近。由于其个体大、肉体肥、口味鲜美等优点，在市场价值方面远高于其它牡蛎类，年产量在130多万吨，产值在60-80亿元。但是，随着养殖规模的不断扩大及养殖环境的恶化，也出现了一些严重问题，养殖牡蛎陆续爆发大规模死亡现象，不但造成了巨大的经济损失，而且严重威胁到了现有产业的可持续发展。
自噬是调节细胞生长，发育的一种重要的程序性细胞死亡方式，它能降解细胞内容物，维持细胞稳态。当细胞面临某些细菌感染时，自噬通过直接包裹胞质内游离的细菌，帮助吞噬体清除其包含的细菌，抵抗细菌毒素等方式保护受感染细胞。
发明内容
本发明的第一个目的是提供来源于香港牡蛎的自噬蛋白beclin-1。
本发明的自噬蛋白beclin-1，其氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供编码上述自噬蛋白beclin-1的Beclin-1基因。
所述的Beclin-1基因的核苷酸序列如SEQ ID NO.1所示。
本发明的第三个目的是提供抑制或干扰Beclin-1基因表达的制剂在阻碍香港牡蛎自噬体形成、抑制NF-κB信号通路、快速响应溶藻弧菌、溶血葡萄球菌的感染的制剂中的应用。
本发明的第四个目的是提供Beclin-1基因的表达量作为牡蛎疾病预防警戒因子的应用。
本发明的第五个目的是提供检测Beclin-1基因的表达量制剂在制备预防牡蛎疾病警戒制剂中的应用。
本发明首次从香港牡蛎中鉴定获得Beclin-1基因，干扰Beclin-1的表达可以抑制自噬体的形成，抑制NF-κB信号通路，而且可以快速响应溶藻弧菌、溶血葡萄球菌的感染。本发明中的Beclin-1在香港牡蛎免疫防御中发挥重要的作用，为牡蛎及其它海水养殖贝类的病害防治、抗病育种提供应用指导。
附图说明
图1.Beclin-1抑制NF-κB的活性；
图2.细菌刺激后Beclin-1的表达模式；
图3.干扰Beclin-1表达阻碍自噬体的形成。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：香港牡蛎Beclin-1基因全长的克隆
1、RNA提取
总RNA的提取使用Trizol(Invitorgen)提取，具体步骤如下：(1)取50mg香港牡蛎组织于液氮预冷的研钵中，将组织磨成粉末之后，转移到装有1ml Trizol的离心管中，吹打、混匀；(2)加入200μL氯仿，剧烈震荡40s，室温放置5min；(3)4℃，12,000×g离心10min，吸取上清转移至新管；(4)加入0.5ml的异丙醇，混匀，-80℃沉淀过夜；(5)4℃，12,000×g离心10min，弃上清；(6)75％乙醇清洗两次沉淀；(7)弃上清，加入50μLDEPC水溶解RNA。
2.香港牡蛎Beclin-1基因的克隆
根据已有的转录组数据，筛选到Beclin-1部分序列，采用SMART RACE cDNAAmpilication Kit试剂盒，通过巢式PCR进行5′和3″末端的扩增获得基因全长，第1轮PCR反应条件：94℃3min；94℃30s，72℃3min，5个循环；94℃30s，70℃30s，72℃3min，5个循环；94℃30s，68℃30s，72℃3min，30个循环；72℃10min，4℃保存。以第1轮PCR产物为模板，进行第2轮PCR反应，反应条件为94℃3min；94℃30s，68℃30s，72℃3min，30个循环；72℃10min，4℃保存。PCR产物用1.5％的琼脂糖凝胶电泳检测，切胶回收目的片段，并克隆至pMD-19T克隆载体，对阳性克隆进行测序获取序列信息。获得Beclin-1基因ORF的核苷酸序列如SEQ IDNO.1所示，为1329bp，编码了442个氨基酸，其氨基酸序列如SEQ ID NO.2所示。
实施例2：香港牡蛎Beclin-1基因的真核表达载体的构建和荧光素酶实验
1.香港牡蛎Beclin-1基因真核表达载体的构建：(1)设计一对涵盖ChBeclin-1ORF的引物F1：CGTAGGATCCATGGCAACCATCAAGGTTGA；R1:GATCGTCGACTCACTTGTTGGCGAACTGAG，引物的两端分别加入BamH-I和SalI位点和保护碱基；(2)使用上述引物进行PCR扩增；(3)PCR产物和pCMV-N-Flag载体分别使用BamH-I和SalI进行双酶切，并回收纯化酶切产物；(4)连接酶切后的PCR片段和pCMV-N-Flag载体；(5)转化，挑取阳性克隆，测序验证载体正确无误，进行后续试验，由此获得Beclin1-Flag。以空载体pCMV-N-Flag作为对照。
2.细胞转染和荧光素酶实验
将HEK293细胞接种于48孔板(1×105个细胞/孔)，脂质体共转染表达载体(Beclin1-Flag)和NF-κB报告基因载体(NF-κB-luc)48h后按照luciferase assay system试剂盒(Promega)的步骤进行，分成4组进行，a、NF-κB-luc 0.1μg+pCMV-N-Flag 0.5μg；b、NF-κB-luc0.1μg+pCMV-N-Flag 0.4μg+Beclin1-Flag 0.1μg；c、NF-κB-luc 0.1μg+pCMV-N-Flag 0.3μg+Beclin1-Flag 0.2μg；d、NF-κB-luc 0.1μg+pCMV-N-Flag 0.1μg+Beclin1-Flag 0.4μg。细胞裂解和荧光的检测简述如下：细胞用PBS洗2次后，加入裂解液100μl/孔，旋转24孔板使裂解液完全覆盖所有细胞，并用细胞刮迅速刮下细胞，转移至1.5ml的微量离心管，并在12000rpm,4℃离心15s，取上清20μl于-80℃暂时保存。测量前冰上解冻，同时将荧光显色剂液放置冰上解冻，打开荧光测量仪，设置合适的参数，将荧光显色剂按照100μl/管的比例加到细胞裂解液中，并迅速置于荧光测量仪内，记录数据。实验共重复三次，每次重复三个。结果表明，香港牡蛎Beclin-1显著抑制NF-κB的荧光素酶的活性(图1)。
实施例3：香港牡蛎Beclin-1在细菌感染下的表达模式
利用实时荧光定量方法(qPCR)检测Beclin-1基因在香港牡蛎受到细菌感染下的表达模式。香港牡蛎200只平均分为两组(四组)，将溶藻弧菌(Vibro alginolyticus)和金黄色葡萄球菌(Staphylococcus haemolyticus)重悬浮在PBS缓冲液中，使得终浓度为1×109cfu/mL，用1mL注射器，注射0.1mL上述浓度弧菌菌液到香港牡蛎闭壳肌，同时对照组每只注射0.1mL PBS缓冲液，空白组不做任何处理。刺激后3、6、12、24、48和72h每组随机取5只牡蛎，抽取血淋巴，800×g离心5min收集血淋巴细胞。采用
Figure BDA0002765374750000041
Premix Ex TaqTM(TaKaRa)染料，EF1α基因做为内参基因(引物：F2:GCTCCACCCAACATCACCACTG；R2:ACGGATTTCCTTTACGGACACG)，Beclin-1定量引物为F3:GGGCTTGGGGTCAGACTGTCTT R3:AAGAAGCGGAATCCACCAGA，使用LightCycler 480II荧光定量PCR仪进行定量分析，PCR反应程序：50℃2min，95℃2min；95℃15s，60℃30s，72℃30s，40个循环；熔解曲线程序：95℃15s，60℃30s，95℃15s。结果显示，ChBeclin-1(即Beclin-1基因)在受到细菌感染后，淋巴细胞中的转录水平显著性的上调，暗示了它参与了牡蛎的免疫反应(附图2)。
实施例4：干扰香港牡蛎Beclin-1表达可抑制自噬体的形成
以香港牡蛎的cDNA为模版，用5′端带T7启动子的ChBeclin-1引物(F4:TAATACGACTCACTATAGGTACTCGAGAACAGGAGCACACTGAG；R3:TAATACGACTCACTATAGGTACTCTCACTTGTTGGCGAACTGAG)和EGFP引物(F5:TAATACGACTCACTATAGGGCAGTGCTTCAGCCGCTACC；R5:CCCTATAGTGAGTCGTATTATTACTTGTACAGCTCGTCCA)进行扩增，PCR扩增片段连接pMD19-T载体并转化大肠杆菌DH5α，挑取阳性克隆作为体外转录模板，利用T7体外转录试剂盒，按照试剂盒提供步骤进行操作，分别获取ChBeclin-1和EGFP dsRNA。将体外转录合成的dsRNA溶解于灭菌的PBS缓冲液中，使其终浓度为1μg/μL。从暂养1周后的香港牡蛎中选取健康个体，随机分为3组，分别标记为PBS对照组、EGFP干扰对照组(dsGFP)和Beclin-1干扰组(ds Beclin-1)，每组在闭壳肌中分别注射100μg ChBelin-1dsRNA，EGFP dsRNA或PBS，然后单独置于一个养殖桶中。注射后72h进行样品的取样，提取总RNA，用于检测目的基因表达量(附图3A)。同时取上述三组外套膜样品，固定于3％戊二醛溶液中，超薄切片用醋酸铀酰/柠檬酸铅染色，使用透射电镜检测自噬体。结果表明，Beclin-1干扰组Beclin-1的表达量显著降低，透射电镜显示自噬体的形成受阻(附图3B)。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种自噬蛋白beclin-1及其编码基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 1329
<212> DNA
<213> 香港牡蛎(Crassostrea hongkongensis)
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cagcttgata accagttaaa gatcacagaa gatgaatgca aggattacag agaattcctg 480
gaaaatttgg acagcgttca tacagaagaa gatgggtcta atttagatgc ggaattgcag 540
cagcttcagg cagaggaaca gcccttgaga caacagttac aaaacctaga gagagaacag 600
gagcacactg aggctctgtt agagaaggag agagggatca gtcaaaaact tgaggatgag 660
gaagataaat attggaaaga atacaatgaa tataaaagac aagtccagga attagaagat 720
gaacaaagga gtgttgacaa ccaacttaaa tatgcacaaa cacaactgga caaactaaag 780
aagacaaatg tgttcaatac cacatttcat atctggcata gtggtcactt tggaaccata 840
aataatttcc gtttgggaag attaccaagt gtcccagttg actggaatga aataaatgct 900
gcttggggtc agactgtctt acttttgaac tcacttgcca aaaagatgaa tttaaccttt 960
cagaggtacc gcctcgttcc ctttggaaac cattcttata ttgagtccct gtccgacaaa 1020
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Phe Val Cys Gln Arg Cys Arg Gln Pro Leu Lys Leu Asp His Ser Phe
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Asn Thr Leu Asp Arg Gln Leu Leu Ala Glu Leu Ser Ala Pro Phe Thr
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Ala Val Glu Ser Asn Glu Asp Glu Leu Asp Ile Ile Ser Lys Pro Ala
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Leu Asp Asp Val Glu Val Asp Glu Asn Tyr Ser Lys Arg Asp Ile Thr
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Ser Thr Pro Glu Pro Asp Glu Asp Ala Gly Asp Phe Leu Leu Leu Gly
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Glu Thr Ser Pro Gly Asn Met Asp Asn Leu Ser His Arg Ile Arg Val
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Ser Ser Ala Leu Phe Asp Val Met Ser Gly Gln Ser Glu Ile Asp His
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Pro Leu Cys Glu Glu Cys Thr Asp Asn Leu Leu Asp Gln Leu Asp Asn
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Gln Leu Lys Ile Thr Glu Asp Glu Cys Lys Asp Tyr Arg Glu Phe Leu
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Glu Asn Leu Asp Ser Val His Thr Glu Glu Asp Gly Ser Asn Leu Asp
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Ala Glu Leu Gln Gln Leu Gln Ala Glu Glu Gln Pro Leu Arg Gln Gln
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Leu Gln Asn Leu Glu Arg Glu Gln Glu His Thr Glu Ala Leu Leu Glu
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Lys Glu Arg Gly Ile Ser Gln Lys Leu Glu Asp Glu Glu Asp Lys Tyr
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Trp Lys Glu Tyr Asn Glu Tyr Lys Arg Gln Val Gln Glu Leu Glu Asp
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Glu Gln Arg Ser Val Asp Asn Gln Leu Lys Tyr Ala Gln Thr Gln Leu
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Asp Lys Leu Lys Lys Thr Asn Val Phe Asn Thr Thr Phe His Ile Trp
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His Ser Gly His Phe Gly Thr Ile Asn Asn Phe Arg Leu Gly Arg Leu
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Pro Ser Val Pro Val Asp Trp Asn Glu Ile Asn Ala Ala Trp Gly Gln
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Thr Val Leu Leu Leu Asn Ser Leu Ala Lys Lys Met Asn Leu Thr Phe
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Leu Ser Asp Lys Ser Lys Glu Leu Pro Leu Tyr Gly Ser Gly Gly Phe
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Arg Phe Phe Trp Asp Thr Lys Phe Asp Gln Ala Met Val Ala Phe Leu
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Asp Cys Leu Gln Gln Phe Lys Glu Glu Val Glu Lys Gly Asp Thr Gly
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Phe Cys Leu Pro Tyr Lys Met Glu Lys Gly Lys Ile Glu Asp Ser Ser
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Thr Gly Thr Ser Tyr Ser Ile Lys Ile Gln Phe Asn Ser Glu Glu Gln
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Trp Thr Lys Ala Leu Lys Tyr Met Leu Thr Asn Leu Lys Trp Gly Leu
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Ala Trp Val Ser Ser Gln Phe Ala Asn Lys
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Claims (6)

1.自噬蛋白beclin-1，其特征在于，氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述的自噬蛋白beclin-1的Beclin-1基因。
3.根据权利要求2所述的Beclin-1基因，其特征在于，所述的Beclin-1基因的核苷酸序列如SEQ ID NO.1所示。
4.抑制或干扰权利要求2或3所述的Beclin-1基因表达的制剂在阻碍香港牡蛎自噬体形成、抑制NF-κB信号通路、快速响应溶藻弧菌、溶血葡萄球菌的感染的制剂中的应用。
5.权利要求2或3所述的Beclin-1基因的表达量作为牡蛎疾病预防警戒因子的应用。
6.检测权利要求2或3所述的Beclin-1基因的表达量制剂在制备预防牡蛎疾病警戒制剂中的应用。
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