CN108610406B - 一种牡蛎细胞凋亡基因caspase 3基因及其在制备病理检测诊断试剂中的应用 - Google Patents

一种牡蛎细胞凋亡基因caspase 3基因及其在制备病理检测诊断试剂中的应用 Download PDF

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CN108610406B
CN108610406B CN201810301750.0A CN201810301750A CN108610406B CN 108610406 B CN108610406 B CN 108610406B CN 201810301750 A CN201810301750 A CN 201810301750A CN 108610406 B CN108610406 B CN 108610406B
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向志明
秦艳平
马海涛
李军
张扬
肖述
张跃环
喻子牛
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Abstract

本发明公开一种牡蛎细胞凋亡基因caspase 3基因及其在制备病理检测诊断试剂中的应用。本发明人发现了一个香港巨牡蛎细胞凋亡的标志性基因‑caspase 3基因，其核苷酸序列如SEQ ID NO.1所示，其在溶藻弧菌(Vibrio alginolyticus)、葡萄球菌(Staphylococcus haemolyticus)感染后显著上调，可以作为检测牡蛎细胞凋亡情况的标准，对于牡蛎疾病的预防提供一个积极而准确的警戒信号，防止了灾害的扩大。该技术的应用，将进一步提升养殖贝类的种质创新能力，支撑产业的高效可持续性发展。

Description

一种牡蛎细胞凋亡基因caspase 3基因及其在制备病理检测 诊断试剂中的应用
技术领域：
本发明涉及贝类免疫与细胞凋亡的分子机理，更具体涉及一种从弧菌刺激后的香港巨牡蛎(Crassostrea hongkongensis)血细胞差减杂交文库中筛选出来的caspase 3基因的核苷酸序列、氨基酸序列及其制备方法和该基因的用途。
背景技术：
我国牡蛎年产量占世界总产量的80％以上，是世界上最大的牡蛎养殖国。香港牡蛎广泛的分布于广西至福建等地方的沿海海域，是华南沿海最重要的牡蛎养殖品种。由于其养殖方式以传统养殖为主，存在着养殖面积广、能耗高等局限性，其产量也因各种条件变化而波动。随着海洋环境污染越来越严重，牡蛎养殖的风险也越来越大。我国目前关于牡蛎病害检测的技术非常落后，基本属于空白状态，因此，提高牡蛎健康监测技术，及时防范病害的传播，是发展和提高牡蛎养殖产业有效手段。
细胞凋亡是多细胞生物正常发育、分化过程中进行细胞删除的一种基本机制，与组织自稳、衰老及细胞损伤密切相关。一旦机理受到病害、环境胁迫时，细胞凋亡途径就会启动，一系列的蛋白酶就会被激活，使细胞有序的被降解掉。在此上述过程中，有一类酶在这个过程中起了关键性作用，它们的结构特征与半胱氨酸蛋白酶很相似，能够特异性地切割Asp后的肽键，故命名为caspase。Caspase家族有很多成员，其中caspase 3是细胞凋亡关键的执行酶，通常是细胞凋亡的是否进行的标志性基因，在生物病理检测中具有重要的应用价值。
发明内容：
本发明的第一个目的是提供一种香港牡蛎细胞凋亡相关的基因序列-caspase 3基因。
本发明的香港牡蛎细胞凋亡相关的基因序列-caspase 3基因，其核苷酸序列如SEQ ID NO.1所示，其编码296个氨基酸，氨基酸序列如SEQ ID NO.2所示。
本发明在香港巨牡蛎转录组测序的基础上，发现了该物种细胞凋亡的标志性基因caspase 3基因。通过RACE技术，我们获得了该基因的全长，其核苷酸序列如SEQ ID NO.1所示，该基因编码的氨基酸序列如SEQ ID NO.2所示。定量PCR实验表明，caspase 3基因在溶藻弧菌(Vibrio alginolyticus)、葡萄球菌(Staphylococcus haemolyticus)感染后显著上调，可以作为检测牡蛎细胞凋亡情况的标准，对于牡蛎疾病的预防提供一个积极而准确的警戒信号，防止了灾害的扩大。
因此，本发明的第二个目的是提供检测caspase 3基因的表达量的检测试剂在制备牡蛎疾病预防警戒试剂中的应用。
所述的牡蛎疾病预防警戒试剂是检测牡蛎细胞凋亡情况的试剂。
所述的牡蛎是香港巨牡蛎。
所述的检测牡蛎细胞凋亡情况的试剂优选是由于溶藻弧菌和/或葡萄球菌感染牡蛎后检测牡蛎细胞凋亡情况的检测试剂。
本发明的第三个目的是提供一种上述caspase 3基因的检测引物；
针对caspase 3基因：
Chcaspase3-F:5'-GGAGCGGTTTCAGGACTTGG-3'
Chcaspase3-R:5'-AGCATTCCGTCGGTAGCGTA-3'。
本发明的第四个目的是提供上述caspase 3基因的检测引物在制备检测caspase3基因的表达量试剂中的应用。
优选，还包括有内参基因GAPDH的检测引物，具体为：
ChGAPDH-F:5'-GGATTGGCGTGGTGGTAGAG-3'
ChGAPDH-R:5'-GTATGATGCCCCTTTGTTGAGTC-3'。
本发明的第五个目的是提供一种caspase 3基因的定量检测方法，其特征在于，是以牡蛎总RNA的反转录产物为模板，以上述caspase 3基因的检测引物为引物，利用定量PCR检测caspase 3基因的表达量。
优选，还利用基因GAPDH作为内参。
优选，所述的定量PCR的反应体系为：
Figure BDA0001619971320000031
反应程序为：95℃5min，1个循环；95℃10s，58.5℃10s，72℃20s，45个循环。
本发明的第六个目的是提供上述caspase 3基因作为牡蛎细胞凋亡标志性基因在制备检测牡蛎细胞凋亡情况的试剂中的应用。
本发明人发现了一个香港巨牡蛎细胞凋亡的标志性基因-caspase 3基因，其在溶藻弧菌(Vibrio alginolyticus)、葡萄球菌(Staphylococcus haemolyticus)感染后显著上调，可以作为检测牡蛎细胞凋亡情况的标准，对于牡蛎疾病的预防提供一个积极而准确的警戒信号，防止了灾害的扩大。该技术的应用，将进一步提升养殖贝类的种质创新能力，支撑产业的高效可持续性发展。
附图说明：
图1是caspase 3蛋白的组织分布，实时定量PCR实验结果表明caspase 3蛋白在各个组织中均有表达，在心脏中含量最低，而血细胞中表达量最高。不同小写字母表示样品间差异显著。
图2是溶藻弧菌刺激诱导caspase 3基因的表达。病原菌刺激后，血细胞中caspase3基因的表达显著上调。星号(**)指示和对照组差异极其显著。
图3是葡萄球菌刺激诱导caspase 3基因的表达。病原菌刺激后，血细胞中caspase3基因的表达显著上调。星号(**)指示和对照组差异极其显著。
具体实施方式：
以下实施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：
1.RNA提取及反转录
a).香港牡蛎细胞或组织加Trizol后，采用组织研磨器研磨，室温放置5min，使其充***解。
b).12,000rpm离心5min，弃沉淀。
c).按200ul氯仿/ml Trizol加入氯仿，振荡混匀后室温放置15min。
d).4℃12,000g离心15min。
e).吸取上层水相，至另一离心管中。
f).按0.5ml异丙醇/ml Trizol加入异丙醇混匀，室温放置5-10min。
g).4℃12,000g离心10min，弃上清，RNA沉于管底。
h).按1ml 75％乙醇/ml Trizol加入75％乙醇，温和振荡离心管，悬浮沉淀。
i).4℃8,000g离心5min，尽量弃上清。
j).室温晾干或真空干燥5-10min。
k).可用50ul H2O，TE buffer或0.5％SDS溶解RNA样品，55-60℃,5-10min。
l).在室温孵育15min之后，加入250μLDNase Stop Solution(DSA)，在13,000×g离心1min。
m).重复步骤a-j,加100μL无核酸酶水，然后，在13,000×g离心1min，弃离心柱，使用分光光度计测量所收集的溶液中的RNA的浓度，分装后，贮存于-80℃。
n).第一条链cDNA的合成
1)DNase I消化
按照如下组分分别将各组的RNA加入到PCR离心管中
RNA 1μg
10XDNase I buffer 1.2μL
DNase I 1μL
Sterile H<sub>2</sub>O Up to 12μL
2)用枪头轻轻吹打混匀并稍作离心；37℃，15min,消化RNA中的DNA
3)加入1μL EDTA混匀，65℃，10min使DNase I失活
4)按照如下组分依次加到离心管：
Figure BDA0001619971320000051
Figure BDA0001619971320000061
用枪头轻轻吹打混匀并稍作离心，放置于37℃，30min；
5)85℃，5s使反转录酶失活，然后4℃短期保存，-20℃长期保存。
2、差减杂交文库构建
差减杂交文库使用PCR-Select cDNA Subtraction Kit(Clontech,USA)试剂盒进行构建。香港巨牡蛎受弧菌刺激8h后，收集血细胞，按照上述的方法提取血细胞总RNA，并使用同样的方法提取对照组(未受弧菌处理)血细胞总RNA。以刺激后香港巨牡蛎作为tester，对照组作为driver，进行扣除杂交。将差减杂交后的cDNAs亚克隆到pGEM-Teasy vector(Promega,USA)并转化到Escherichia coli JM109(Promega,USA)感受态细胞。随机挑选2000个克隆并进行测序。
3.caspase 3基因全长的克隆
依据步骤2所得到的克隆测序，通过BLASTEN等生物软件分析并确定caspase 3序列，再以此序列为模版，通过GeneRacerTM RACE Ready cDNA试剂盒(Invitrogen,USA)进行该基因的5'/3'RACE，具体操作参照说明书。得到RACE序列后，通过拼接，获得该基因的全长cDNA,分析其编码区，其编码区的核苷酸序列如SEQ ID NO.1所示，命名为caspase 3基因，其编码的氨基酸序列如SEQ ID NO.2所示，命名为caspase 3蛋白。
4.实时定量PCR
进行实时定量PCR所使用的试剂盒为LightCycler 480SYBR Green I(Roch)，所使用的仪器为LightCycler 480System(Roche)，所使用的模板为总RNA的反转录产物，所使用的内参GAPDH，所使用的引物有：
针对caspase 3基因：Chcaspase3-F:5'-GGAGCGGTTTCAGGACTTGG-3'
Chcaspase3-R:5'-AGCATTCCGTCGGTAGCGTA-3'
针对内参GAPDH基因：ChGAPDH-F:5'-GGATTGGCGTGGTGGTAGAG-3'
ChGAPDH-R:5'-GTATGATGCCCCTTTGTTGAGTC-3'
qRT-PCR实验采用Light-Cycler 480II System(Roche)进行反应体系如下：
Figure BDA0001619971320000071
热循环条件如下：95℃5min，1个循环；95℃10s，58.5℃10s，72℃20s，45个循环。使用2－ΔΔCT方法对转录本进行相对定量。实验组和对照组均设置3个平行反应。
5.组织分布及样品处理
分别提取5只香港巨牡蛎的各个组织的总RNA(见步骤1)并反转录为cDNA，采用Real-time PCR技术检测了caspase 3基因在香港巨牡蛎的淋巴细胞，腮，触唇，性腺，消化腺，外套膜，围心腔，闭壳肌中的分布情况。结果显示它在各个组织中广泛表达，其中外套膜中表达量最低，在血淋巴表达最高(图1)。
取正常香港巨牡蛎，分别注射1.0×109个活的病原菌，以PBS注射组为参照，在不同的时间点随机取样(5个一组)，提取血细胞的总RNA并反转录为cDNA，见步骤1。以此为模版，进行样品的Real-time PCR检测caspase 3基因的表达量，结果发现溶藻弧菌(图2)，葡萄球菌(图3)感染都能引起血细胞中caspase 3基因的显著上调表达。
序列表
<110> 中国科学院南海海洋研究所
<120> 一种牡蛎细胞凋亡基因caspase 3基因及其在制备病理检测诊断试剂中的应用
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aaaatggaac ttctgcacat gctcactcaa gtcaatcgca tggcggcgta tgaattcgag 780
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Tyr Ser Thr Val Pro Gly Phe Tyr Ser Trp Arg Asn His Gln Glu Gly
210 215 220
Ser Trp Leu Ile Gln Ala Leu Cys Ile Val Leu Glu Asn Tyr Gly Ser
225 230 235 240
Lys Met Glu Leu Leu His Met Leu Thr Gln Val Asn Arg Met Ala Ala
245 250 255
Tyr Glu Phe Glu Ser Cys Ser Asp Glu His Phe Thr Asp Glu Val Lys
260 265 270
Gln Met Pro Cys Ile Val Ser Met Leu Thr Arg Tyr Val Tyr Phe Arg
275 280 285
Pro Lys Lys Pro Asp Ile Arg Asp
290 295

Claims (4)

1.一种检测caspase 3基因的表达量的检测试剂在制备预防警戒溶藻弧菌或葡萄球菌感染牡蛎的试剂中的应用，所述的caspase 3基因，核苷酸序列如SEQ ID NO.1所示，所述的牡蛎是香港巨牡蛎。
2.根据权利要求1所述的应用，其特征在于，所述的试剂是检测牡蛎细胞凋亡情况的试剂。
3.根据权利要求2所述的应用，其特征在于，所述的检测牡蛎细胞凋亡情况的试剂是由于溶藻弧菌和/或葡萄球菌感染牡蛎后检测牡蛎细胞凋亡情况的检测试剂。
4.caspase 3基因作为牡蛎细胞凋亡标志性基因在制备检测香港巨牡蛎在感染溶藻弧菌或葡萄球菌导致的细胞凋亡情况的试剂中的应用，所述的caspase 3基因，核苷酸序列如SEQ ID NO.1所示。
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