CN110055200A - 一株变形假单胞菌clpV基因沉默菌株 - Google Patents
一株变形假单胞菌clpV基因沉默菌株 Download PDFInfo
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Abstract
本发明提供一株变形假单胞菌clpV基因沉默菌株，所述菌株为Pseudomonas plecoglossicida clpV‑RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772。所构建的变形假单胞菌clpV基因稳定沉默菌株对海水鱼的致病力及显著下降，不仅可用于研究变形假单胞菌的致病机理，还可用于研制变形假单胞菌的弱毒疫苗并为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
Description
技术领域
本发明属于微生物技术领域，更具体涉及一株变形假单胞菌clpV基因沉默菌株。
背景技术
变形假单胞菌(Pseudomonas plecoglossicida)是大黄鱼、斜带石斑鱼等海水养殖鱼类“内脏白点病”的病原菌，每年造成的直接经济损失超亿元。
在先前的研究中，与野生型变形假单胞菌（在18℃下培养）的纯培养相比，在感染后24、48、72和96 小时，所有差异表达基因都被用于加权基因共表达网络分析并作为不同的基因共表达模块鉴定。所有模块均用KEGG富集分析。用这种方法，筛选出clpV基因被选择为 在VI 型分泌系统（T6SS）中的一个候选关键毒力基因。
本发明通过比较转录组学分析技术，结合RNA干扰这一定向高效的基因沉默技术，构建一株变形假单胞菌clpV基因稳定沉默菌株，并对该菌株的毒力效果进行检测。所构建的变形假单胞菌clpV基因稳定沉默菌株对海水鱼的致病力及显著下降，不仅可用于研究变形假单胞菌的致病机理，还可用于研制变形假单胞菌的弱毒疫苗并为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
发明内容
本发明的目的在于提供一株变形假单胞菌clpV基因稳定沉默菌株及其功能。
为实现上述目的，本发明采用如下技术方案：
一株变形假单胞菌clpV基因沉默菌株，所述菌株为Pseudomonas plecoglossicidaclpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772，地址为武汉，武汉大学。
本发明提供的这株变形假单胞菌clpV基因稳定沉默菌株是通过下面的技术手段进行构建，
（1）通过比较转录组学分析技术，将筛选致病功能基因目标锁定clpV基因；
（2）合成shRNA，退火后连入pCM130/tac载体，通过电转技术导入变形假单胞菌感受态细胞，构建变形假单胞菌clpV基因稳定沉默菌株；利用QPCR技术，对沉默效果进行检验；最终获得变形假单胞菌clpV基因沉默菌株。所述的pCM130/tac载体是利用质粒pCM130加上启动子tac构成。
（3）利用人工感染实验，研究变形假单胞菌野生株、clpV基因沉默株的毒力，以及clpV基因沉默对变形假单胞菌及斜带石斑鱼基因表达的影响。
通过基因测序和比对，本发明提供的菌株为一株变形假单胞菌clpV基因稳定沉默菌株。
所述clpV基因序列如SEQ ID NO.1所示。
本发明的优点在于：
人工感染实验结果表明，变形假单胞菌clpV基因稳定沉默菌株的毒力显著低于变形假单胞菌野生株的毒力，在同等感染条件下不会引起斜带石斑鱼的死亡，具有研制减毒疫苗的潜力。
Dual RNA-seq分析结果表明，clpV基因的稳定沉默不仅能够显著影响变形假单胞菌的转录组表达，而且也能显著影响斜带石斑鱼的转录组表达，这说明变形假单胞菌clpV基因稳定沉默菌株可用于研究变形假单胞菌的致病机理，还可用于为海水鱼“内脏白点病”的预防和治疗寻找新的靶点。
附图说明
图1为斜带石斑鱼感染变形假单胞菌后的存活率图。
图2为斜带石斑鱼感染后脾脏组织的病症变化图。
图3为clpV基因沉默株和野生株在组织器官分布的差异倍数动态变化图。
具体实施方式
实施例1
本发明实施例提供的变形假单胞菌clpV基因稳定沉默菌株的构建方法包括以下步骤：
S101：通过比较转录组学分析技术，对变形假单胞菌基因表达水平进行检测；利用生物信息学技术，极大的缩小研究范围挑选出最具有研究价值的致病功能基因，用KEGG富集分析这种方法，筛选出clpV基因被选择为 在VI 型分泌系统（T6SS）中的一个候选关键毒力基因。故将目标锁定clpV基因；
S102：利用Thermo-fisher Scientific 公司在线shRNA设计工具（http://rnaidesigner.thermofisher.com/rnaiexpress/setOption.dodesignOption=shrna&pid=708587103220684543），设计并合成shRNA，然后使用退火缓冲液退火形成双链shRNA。shRNA序列F:5’-TGCAGTGTTCAAACGCATTTCCTTCAAGAGAGGAAATGCGTTTGAACACTGCTTTTTTT-3’
R:5’-GTACAAAAAAAGCAGTGTTCAAACGCATTTCCTCTCTTGAAGGAAATGCGTTTGAACACTGCATGCA-3’，从百奥迈科公司购买原始质粒pCM130加上去的启动子tac，构成pCM130/tac质粒。买使用限制性内切酶BsrGI和 NsiI双酶切pCM130/tac质粒使其线性化，然后使用T4连接酶将线性化质粒和双链shRNA连接。将重组质粒先热击转化进大肠杆菌DH5α感受态细胞中，37℃扩大培养，测序成功后提取重组质粒，电击转化进变形假单胞菌感受态细胞中，18℃条件下扩大培养。此菌株即为变形假单胞菌clpV沉默突变株。所述变形假单胞菌clpV沉默突变株为Pseudomonas plecoglossicida clpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772，地址为武汉，武汉大学。
使用qRT-PCR技术，对沉默效果进行检验，上游引物序列（5’-3’’）为：CGAGGTCATTAAAGTTGCCCAATC，下游引物序列（5’-3’’）为：CGCCGAAACCGACATACCCT；检验结果与转录组测序结果一致，感染后clpV基因表达量上调；
S103：利用人工感染实验，对变形假单胞菌野生株和clpV基因沉默株的毒力进行对比。
clpV基因沉默株（clpV-RNAi株）、变形假单胞菌野生株和PBS（NaCl 0.8g、KCl0.02g、Na2HPO4 0.36g、 KH2PO4 0.024g、H2O 1L）分别对三组斜带石斑鱼进行胸腔注射感染，菌株感染浓度为103cfu/g，每条鱼注射0.2 mL，每组20尾鱼，然后继续正常暂养，并记录每天各组鱼的存活状况。
结果发现，对注射clpV-RNAi株和PBS的斜带石斑鱼，在注射后未观察到长达20 天都没观察到死亡。注射含有野生菌株的斜带石斑鱼在注射7 天内死亡(图1)。注射含有野生菌株的鱼的脾脏中观察到大量白色结节，表现出与自然疾病相同的症状。相反，在注射clpV-RNAi株和PBS的鱼中没有观察到白斑点(图2)。此外，所有接受clpV-RNAi菌株的斜带石斑鱼的脾脏、肾脏和肝脏都变得红肿，但在注射5-8天时未能形成可见的结节，肿胀逐渐消失。
变形假单胞菌的看家基因 gyrB 的 DNA 拷贝数被用来评估该病原菌在感染组织中的丰度。把 gyrB基因序列与 NCBI 数据库比对后，筛选出种间特异性高和种内变异率小的序列，使用 Primer Express 3.0 软件进行引物和探针的设计。为了便于与试剂盒配套使用，引物和探针由美国 Life Technologies 公司合成。
dPCR 引物和探针的序列
正向引物 5'-CAGACCTACGTGCACGGTGTT-3'
反向引物 5'-GGATTCACTGTCGCCCACTAC-3'
探针 5'-VIC-AGGCTCCAATGGC-MGB-3'
用dPCR方法检测感染了clpV-RNAi株在斜带石斑鱼的脾脏、肝脏、头肾、体肾和血液中的动态分布。图3显示了clpV-RNAi株和野生株分布的差异倍数动态变化。在感染后的大部分时间点，斜带石斑鱼中的脾脏和血液中的clpV-RNAi菌株丰度明显低于野生菌株。clpV-RNAi株在在感染后72到120小时头肾和体肾中的丰度表现为急剧下降。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 集美大学
<120> 一株变形假单胞菌clpV基因沉默菌株
<130> 8
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 2466
<212> DNA
<213> 人工序列
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cgtatccgcg gcggatcgct gctgttggcg ctggtgtcgc gcgcaagcta ctacaccgcc 120
ggtgctcgtt acagtgcagt gttcaaacgc atttccgtag acgcgttgac cgccgaattc 180
gccgcgctgg tggccagctc aagcgaaggg cccttggaca ttttgcacag tgcggccggg 240
gccgccaacc ctgtcgaggg cgctgtcgca gaaaaccaaa tcgcacgctt ttgcgaagac 300
cttaccgaga aggcccggca ggggaaaatc gaccccgtct tcgggcgcga tgacgaagtg 360
cgtcagatga tcgatattct cgcgcgtcgt cgcaagaaca accctattgc agtgggtgag 420
ccgggtgttg gcaagtcggc agtggtcgag ggcttggcgc tgttgatcaa tgagggcaac 480
gtgccggcgt ttctccaggg cacacgcctg ctcagcctgg atctgggggc acttgaggca 540
ggcgccagcg tcaagggtga gttcgaaaac cgtctgcgtg gtgtcattga ggagatcaag 600
gcctcgacca caccggtggt cttgtttatc gatgaagcac acatgttgat cggcgcaggc 660
ggggcggcgg gtggctcgga tgcggccaac ctgctcaagc ctgcgttggc ccgtggtgaa 720
ctgcgcacca tcgccgcaac aacctggtcg gaatataaaa agtatttcga gaaggacccg 780
gcgctggcca gacgctttca gctggtcaag ctggatgagc ccagtgtcgg cactgcagtg 840
cacattctgc gagggctcaa agggcattat gaaaaggtcc atggggtcag cgtgcgtgat 900
gatgccatcg ttgcggcggc tgagttgtcc gatcgataca tcaccggccg gttgctaccg 960
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aaacctgcgc aactggaacg tctggagcga cagctttcgg gcctgatgcg tgaacgggcg 1080
gcgttggagc gggaccaggc cgacggcttc ccgatcgaac ctcagcgcct ggaaaccata 1140
gccgatgagc aagcgcacat tgagggggct gtggatgctc tggaggcgcg ctgggccaat 1200
gaacggcagg cggcgatgcg ggtgctggag gcgcgcacca acctcgcaac gctcaagcag 1260
gccggtgacg gcagcacagc gctgggctcg ctcgacgcac accagatgga gctcgaacag 1320
gctcgcgatg cactggcgac gttgcagggc gacgagcctt tgctctttac cgaggtcggt 1380
cccgacacga ttgcccaggt ggtctcggac tggacgggga taccgttagg taaggttcag 1440
cgtgatgcct ctgatggcct gctggcgctg gccagccacc tcaagcagcg tatctgcggc 1500
caggaagccg ccgtggagca gattgccgag gtcattaaag ttgcccaatc gggcttgcgt 1560
gaccctcagc agccgttggg ggtcttcttg ctggtcgggc cctcgggagt cggcaagacc 1620
gaaacagcgc tggcgctggc agagcaattg ttcggcggcg agcaggcgtt gattaccgtg 1680
aacatgagcg agtatcaaga aaagcacacc gtcagccgtt tggtcggttc gcctgcaggg 1740
tatgtcggtt tcggcgaggg tggcatgttg accgaagccg tgcgcaaacg cccttattcg 1800
gtcgtgttgc tggatgaagt ggaaaaggcg cacttggaag tagtgaatat tttctaccag 1860
gtattcgaca agggcttttt gacagatggc gaggggcggc agatcgactt cagaaacacc 1920
gtgattctgc tgaccagcaa cttggccagt gatgaaattc aagcgcgttg cgcaggcgca 1980
cgtccagacg cggaagcgct ggtcgagcat gttcgcccac ggctgtctca atacttcaag 2040
cctgcattat tggcgcgaat gacgattgtg ccctatttca ccctggcggg cgcgcaattg 2100
gaagagattg tgcggctgaa gttggcgcgt ctgaccgaac gcctttggtg gacatcgcgg 2160
gtcaacctga gcttcgccga agaggtcgcc acggtcattg ccgagcgctg tacggaagtg 2220
gaaagtggtg cgcgcaatat cgattacatc ttgcgtaaaa gtctcacccc gcggctttcc 2280
gaggccctgt tggcggccat tgcgcagcag cgaccgctgt gcacgctgac ggttgcagtg 2340
agcccgacgg gcgagtggtt gatcacggct gtcgagcgcg tcgatccggt tgttgtggag 2400
gccgacaatg caggcgttgt caccgctagc cctgcagcaa atgcagagac agtgacgccc 2460
gtctga 2466
<210> 2
<211> 59
<212> DNA
<213> 人工序列
<400> 2
tgcagtgttc aaacgcattt ccttcaagag aggaaatgcg tttgaacact gcttttttt 59
<210> 3
<211> 67
<212> DNA
<213> 人工序列
<400> 3
gtacaaaaaa agcagtgttc aaacgcattt cctctcttga aggaaatgcg tttgaacact 60
gcatgca 67
<210> 4
<211> 24
<212> DNA
<213> 人工序列
<400> 4
cgaggtcatt aaagttgccc aatc 24
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
cgccgaaacc gacataccct 20
<210> 6
<211> 21
<212> DNA
<213> 人工序列
<400> 6
cagacctacg tgcacggtgt t 21
<210> 7
<211> 21
<212> DNA
<213> 人工序列
<400> 7
ggattcactg tcgcccacta c 21
<210> 8
<211> 13
<212> DNA
<213> 人工序列
<400> 8
aggctccaat ggc 13
Claims (5)
1.一株变形假单胞菌clpV基因沉默菌株，其特征在于：所述菌株为Pseudomonas plecoglossicida clpV-RNAi，已于2018年11月12日于中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2018772。
2.如权利要求1所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述方法包括如下：
（1）通过比较转录组学分析技术，将筛选致病功能基因目标锁定clpV基因；
（2）合成shRNA，退火后连入pCM130/tac载体，通过电转技术导入变形假单胞菌感受态细胞，构建变形假单胞菌clpV基因稳定沉默菌株；利用QPCR技术，对沉默效果进行检验，最终获得变形假单胞菌clpV基因沉默菌株。
3.根据的权利要求2所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述clpV基因序列如SEQ ID NO.1所示。
4.根据的权利要求2所述的一株变形假单胞菌clpV基因沉默菌株的构建方法，其特征在于：所述的pCM130/tac载体是利用质粒pCM130加上启动子tac构成。
5.如权利要求1所述的一株变形假单胞菌clpV基因沉默菌株在制备预防和治疗海水鱼内脏白点病制剂中的应用。
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454872A (zh) * | 2020-03-16 | 2020-07-28 | 扬州大学 | 禽致病性大肠杆菌VI型分泌系统clpV基因缺失株及其构建方法和应用 |
| CN112063551A (zh) * | 2020-08-31 | 2020-12-11 | 浙江万里学院 | 杀香鱼假单胞菌六型分泌系统缺失突变株及其应用 |
| CN112625996A (zh) * | 2021-02-04 | 2021-04-09 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN112662608A (zh) * | 2021-02-04 | 2021-04-16 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN113151134A (zh) * | 2021-05-12 | 2021-07-23 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113528407A (zh) * | 2021-05-31 | 2021-10-22 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN114703115A (zh) * | 2022-04-22 | 2022-07-05 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN116287330A (zh) * | 2022-12-30 | 2023-06-23 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047783A (zh) * | 2016-05-25 | 2016-10-26 | 浙江万里学院 | 杀香鱼假单胞菌ExoU基因敲除突变株及其应用 |
| CN106834199A (zh) * | 2017-02-27 | 2017-06-13 | 山东省农业科学院畜牧兽医研究所 | 铜绿假单胞菌pyoS5基因敲除突变株及构建方法及应用 |
-
2019
- 2019-01-07 CN CN201910013464.9A patent/CN110055200B/zh not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047783A (zh) * | 2016-05-25 | 2016-10-26 | 浙江万里学院 | 杀香鱼假单胞菌ExoU基因敲除突变株及其应用 |
| CN106834199A (zh) * | 2017-02-27 | 2017-06-13 | 山东省农业科学院畜牧兽医研究所 | 铜绿假单胞菌pyoS5基因敲除突变株及构建方法及应用 |
Non-Patent Citations (2)
| Title |
|---|
| 肖何: "罗尔斯通氏菌中 Reut_A2805 蛋白调节 VI 型分泌系统抗氧化功能的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 胡娇等: "大黄鱼(Pseudosciaena crocea)内脏白点病", 《海洋与湖沼》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454872A (zh) * | 2020-03-16 | 2020-07-28 | 扬州大学 | 禽致病性大肠杆菌VI型分泌系统clpV基因缺失株及其构建方法和应用 |
| CN112063551A (zh) * | 2020-08-31 | 2020-12-11 | 浙江万里学院 | 杀香鱼假单胞菌六型分泌系统缺失突变株及其应用 |
| CN112662608B (zh) * | 2021-02-04 | 2022-06-10 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN112662608A (zh) * | 2021-02-04 | 2021-04-16 | 集美大学 | 一株变形假单胞菌exbB基因稳定沉默菌株及应用 |
| CN112625996A (zh) * | 2021-02-04 | 2021-04-09 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN112625996B (zh) * | 2021-02-04 | 2022-06-10 | 集美大学 | 一株变形假单胞菌znuA基因稳定沉默菌株及应用 |
| CN113151134A (zh) * | 2021-05-12 | 2021-07-23 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113151134B (zh) * | 2021-05-12 | 2023-09-12 | 集美大学 | 一株变形假单胞菌fliG基因沉默菌株及其应用 |
| CN113528407A (zh) * | 2021-05-31 | 2021-10-22 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN113528407B (zh) * | 2021-05-31 | 2023-01-10 | 集美大学 | 一株变形假单胞菌tonB基因沉默菌株及其应用 |
| CN114703115A (zh) * | 2022-04-22 | 2022-07-05 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN114703115B (zh) * | 2022-04-22 | 2023-09-29 | 集美大学 | 一种变形假单胞菌fliS基因沉默菌株和用途 |
| CN116287330A (zh) * | 2022-12-30 | 2023-06-23 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
| CN116287330B (zh) * | 2022-12-30 | 2023-12-05 | 中国科学院生态环境研究中心 | 一种可检测多种毒力基因的高通量实时荧光定量pcr芯片及其检测方法 |
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