CN112301000A - 一种能将应激信号转变为egfp荧光信号的报告细胞系 - Google Patents
一种能将应激信号转变为egfp荧光信号的报告细胞系 Download PDFInfo
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Abstract
本发明公开了一种能将应激信号转变为EGFP荧光信号的报告细胞系，同时还公开了如何制备该报告细胞系相关的方法，以猪基因组中的一个能识别特异性应激源的猪HSPA6基因为前提，利用CRISPR/Cas9介导基因敲入技术，成功地构建了猪HSPA6基因与EGFP同步表达的PK‑15细胞系，结果显示该细胞系能高效且灵敏的将猪HSPA6基因应激源对该细胞系的刺激信号转变为EGFP荧光信号；本发明可以应用于病原检测、环境监测、食品安全、药物及毒物研究等方面。
Description
技术领域
本发明公开了一种能将应激信号转变为EGFP荧光信号的报告细胞系，同时还公开了如何制备该报告细胞系相关的方法，属于生物技术领域。
技术背景
作为一项新兴技术，细胞传感技术的应用领域十分广泛。其敏感性和快捷性等特点使其在病原检测、环境监测、食品安全、药物及毒物研究等方面也具有十分重要的应用价值和科研价值。寻找具有灵敏感应特性的细胞或基因,并加以合理的利用和改造，是细胞传感技术最关键的环节。
HSPA6属于诱导型HSP70家族的一员，该基因属于严格诱导表达型基因，正常条件下，HSPA6基因基本不表达，但被特定应激源刺激后，该基因会迅速且大量的表达。HSPA6基因的表达特性被认为在细胞传感研究领域具有很大的潜在研究价值。
发明内容
本发明的目的是提供一种能将应激信号转变为EGFP荧光信号的报告细胞系，该细胞系能用于特异识别HSPA6基因的应激源并将应激源对该细胞系的刺激信号转变为EGFP荧光信号。
本发明还提供了一种能有效将应激信号转变为EGFP荧光信号的报告细胞系的制备方法，以CRISPR/Cas9介导的定点整合技术，在猪源细胞系中实现EGFP基因与HSPA6基因同步表达的可行性，并进一步明确各共表达细胞系中EGFP基因与HSPA6基因表达的同步性。
本发明提供的一种能有效将应激信号转变为EGFP荧光信号的报告细胞系，其基因序列如SEQ no.1所示：
该报告细胞系能高效且快速地将HSPA6基因应激源对PK-15细胞的刺激信号转变为EGFP荧光信号。
本发明提供的一种能有效将应激信号转变为EGFP荧光信号的报告细胞系的制备方法，包括以下步骤：
1）猪HSPA6基因特异sgRNA的设计和筛选
a. sgRNA的设计，根据猪HSPA6基因碳端的DNA序列，选择合适的PAM序列（NGG）用于基因编辑sgRNA靶点的设计，所设计的sgRNA序列如下：
SgRNA-1序列：5- CATTGAGGAGGTTGATTAAA-3；
SgRNA-1作用位点的序列：5- TTTAATCAACCTCCTCAATG-3；
SgRNA-2序列：5- TAATCAACCTCCTCAATGAC-3；
SgRNA-2作用位点的序列：5- GTCATTGAGGAGGTTGATTA-3；
SgRNA-3序列：5- GTCACAGCTGACTTGTCAGG-3；
SgRNA-3作用位点的序列：5- CCTGACAAGTCAGCTGTGAC-3；
b.高效sgRNA的筛选，构建含上述sgRNA的表达载体，然后电转入细胞，通过测序等方法筛选出切割效率高且潜在脱靶效率低的sgRNA；
2）根据靶位点的序列，设计并合成EGFP同源打靶载体
根据上述筛选出的sgRNA，结合该sgRNA对HSPA6基因的的精确切割位点，设计设计并合成EGFP同源打靶载体；该打靶载体的主要原件依次为：上游同源臂、p2A位点、EGFP基因、下游同源臂及原核表达的骨架载体；
3）对所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的筛选和鉴定
a. sgRNA表达质粒和EGFP同源打靶载体的共转染；
b.极限稀释细胞铺板；
c. 细胞克隆的挑取和鉴定；
4）对所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的荧光报告功能分析及验证；
a.HSPA6应激源对上述EGFP荧光报告细胞系的刺激实验；
b. EGFP荧光报告细胞系的报告能力评估。
本发明所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在制备HSPA6基因特异的活细胞传感器中的用途。
本发明所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在基因互作、启动子活性区域分析、病原检测、环境监测、食品安全、药物及毒物研究等方面的应用。
本发明所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在制备HSAP6基因相同位点制备荧光报告动物模型中的应用。
本发明所述的一种能将应激信号转变为EGFP荧光信号的报告细胞系在猪HSPA6应激源检测和筛选领域的应用。
HSPA6属于诱导型HSP70家族的一员，该基因属于严格诱导表达型基因，其表达特性被认为在细胞传感研究领域具有很大的潜在研究价值。本发明以CRISPR/Cas9介导的定点整合技术探究猪源细胞系中实现EGFP基因与HSPA6基因同步表达的可行性，并进一步明确各共表达细胞系中EGFP基因与HSPA6基因表达的同步性。
本发明的积极效果在于：
公开了一种能将应激信号转变为EGFP荧光信号的报告细胞系，以猪基因组中的一个能识别特异性应激源的猪HSPA6基因为前提，利用CRISPR/Cas9 介导基因敲入技术，成功地构建了猪HSPA6基因与EGFP同步表达的PK-15细胞系，结果显示该细胞系能高效且灵敏的将猪HSPA6基因应激源对该细胞系的刺激信号转变为EGFP荧光信号；本发明可以应用于病原检测、环境监测、食品安全、药物及毒物研究等方面。
附图说明：
图1为本发明用于评估3条不同sgRNA切割效率的测序峰图；
图2为本发明EGFP定点打靶质粒载体示意图；
图3为本发明EGFP定点整合细胞克隆的荧光显微镜图；
图4为本发明EGFP定点整合的细胞克隆的PCR鉴定电泳图；
图5为本发明EGFP报告细胞克隆对特定应激源的荧光报告能力测试结果。
具体实施方式
通过以下实施例进一步举例描述本发明，并不以任何方式限制本发明，在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
1、靶向猪HSPA6基因sgRNA序列的设计及表达载体的构建
设计并合成了3条针对猪HSPA6位点的sgRNA序列。上述设计完成的shRNA序列合成；6条单链的sgRNA的DNA序列分别经过退火后形成3条靶向猪HSPA6碳端不同位点的sgRNA的寡核苷酸链；然后将该寡聚核苷酸连连入PX330质粒载体。
这3条sgRNA的序列及其作用位点的序列分别为：
SgRNA-1序列：5- CATTGAGGAGGTTGATTAAA-3；
SgRNA-1作用位点的序列：5- TTTAATCAACCTCCTCAATG-3；
SgRNA-2序列：5- TAATCAACCTCCTCAATGAC-3；
SgRNA-2作用位点的序列：5- GTCATTGAGGAGGTTGATTA-3；
SgRNA-3序列：5- GTCACAGCTGACTTGTCAGG-3；
SgRNA-3作用位点的序列：5- CCTGACAAGTCAGCTGTGAC-3；
2、高效sgRNA的评估和筛选
通过对3种sgRNA的表达载体进一步的测序后，进行质粒的大提和质粒的乙醇沉淀，将纯化后一定浓度的三种sgRNA的PX330的表达载体，通过电穿孔转染的方式引入到PK-15细胞中，转染后的72小时，提取各组细胞的基因组，然后用特异性的检测突变效率的引物进行pcr反应，将所获得的PCR产物一方面送去测序通过对测序峰图的分析初步评估个sgRNA的切割效率，同时将剩下的PCR产物用于连T载体或同过T7E1分析来精确的评估各sgRNA对靶基因的切割效率，参见图1。
实施例2
EGFP定点整合打靶载体的构建
根据筛选出的高效sgRNA设计并构建与该sgRNA配套的EGFP基因打靶载体（EGFP-KI-Donor），该打靶载体的主要原件依次为：上游同源臂、p2A位点、EGFP基因、下游同源臂及原核表达的骨架载体。该EGFP定点整合打靶质粒与筛选出的sgRNA共同作用可以对猪基因组的HSPA6基因的C端进行特异的基因改造，改造后的基因序列参见SEQ no.1。然后再结合荧光显微镜与PCR的方法可以很方便的分析外源基因在该sgRNA识别位点整合并表达的可行性，参见图2所示。
实施例3
PX330质粒与EGFP定点整合打靶载体的共转染
复苏PK-15细胞细胞，传至F3代时，消化F3代的PK-15细胞，用DPBS洗2~3遍后，弃上清，加入电转染缓冲液，然后将PX330质粒与EGFP-KI-Donor质粒按比例加入到细胞和缓冲液中，用移液器轻轻混匀后，轻轻的将混合液移入电极杯中，将电极杯放到电穿孔仪器上进行电击操作。电击完成后，将电极杯在4℃静置10分钟后，将电极杯中的混合液转入细胞培养皿中。最后将该细胞培养皿置于37℃二氧化碳培养箱中培养。培养12小时后，换液。通过电转染的方法将表达sgRNA的PX330质粒与EGFP定点整合打靶载体高效地引入到了PK-15细胞中。
实施例4
EGFP报告细胞克隆的挑取与鉴定及荧光显微镜成像图
电转染72h后，通过极限稀释的方法将PK-15细胞铺到100mm细胞培养皿中，2~3天更换一次细胞培养液。9~10天后待细胞克隆长成后，将细胞放在42℃的细胞培养箱中进行热刺激处理1h，然后放在37℃的培养箱中适应1h后，接着在荧光显微镜下将发绿光的细胞克隆统一做上标记，然后将这些标记过的克隆挑取入24孔细胞培养板中接着培养。2~3天后，待24孔板中的细胞长至一定得汇合度，对细胞进行传代同时分出部分克隆的细胞，将这些细胞用NP40裂解液裂解后再通过PCR和测序的方法进一步验证定点EGFP的整合事件。参见图3、图4所示。成功地筛选获得了EGFP报告细胞系，并证明了这些筛选出的EGFP报告细胞系能有效的将热应激信号转变为EGFP的荧光信号。
实施例5
EGFP报告细胞克隆对特定应激源的的荧光报告能力测试
如图5所示，将高温（42℃）、MG-132、姜黄素、Zn2+等引入到该EGFP报告细胞的培养环境中，进行刺激实验，刺激实验的结果表明，该EGFP报告细胞系能高效且灵敏地将以上刺激源对猪HSPA6的刺激信号，转变为EGFP荧光信号。参见图5所示。
结论：
本发明以猪基因组中的一个能识别特异性应激源的猪HSPA6基因为前提，利用CRISPR/Cas9 介导基因敲入技术，成功地构建了猪HSPA6基因与EGFP同步表达的PK-15细胞系，结果显示该细胞系能高效且灵敏的将猪HSPA6基因应激源对该细胞系的刺激信号转变为EGFP荧光信号。
序列表
<110> 吉林大学
<120> 一种能将应激信号转变为EGFP荧光信号的报告细胞系
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4609
<212> DNA
<213> 猪(pig)
<400> 1
atgtccgctg caagagaagt ggccataggc atcgacttgg gtaccacata ctcctgcgtg 60
ggtgtgttcc agcacggtcg ggtggagatc ctagcaaatg accagggcaa ccgcaccacc 120
cccagctacg tggccttcac cgacaccgag cggctggtgg gcgatgcagc caagagccag 180
gcggccctga acccgcagaa taccgtgttt gacgccaagc ggctaattgg gcgcaagttt 240
gcagacccca ccgtgcagtc agacttgaag cactggcctt tccaggtggt gagcgagggt 300
gggaagccca aggtgcgcgt ttcctaccgt ggggaggaca aggcgttcta cccggaggag 360
atttcgtcca tggtgctgag caagatgaag gagactgcgg aggcgtacct gggccagccc 420
gtgaggcacg ctgtgatcac cgtgcctgcc tacttcaacg actctcagcg tcaggccacc 480
aaggacgcag gtgccattgc agggctcaac gtgctgagga tcattaatga gcccacagct 540
gctgccatcg cctatggcct ggaccggagg ggcgctggtg agcggaacgt gctcattttc 600
gacttgggtg gaggaacctt cgacgtgtcg gtccttacta ttgacgcagg tgtctttgag 660
gtgaaggcca cggctggaga cactcacctg ggtggagagg acttcgacaa ccgtttggtg 720
aaccacttca atcttggcct ttggcctttg gggcacagga tgccttgtac tccaggcatt 780
tattgcacac ctgagttctg ggtttgagct gggaacccgt ccaggcctca gaaagaaaac 840
acacactatg cctttccaac tcatcccttg gtactatgct cccaggagat atgagcccgc 900
aggggctgga taagagaagg gacattctca ccacagttca cctgcatttt ccttcctcct 960
aggctgttcc caagccacag ctccaggttc tggctccttt ctgggctgcg acttgataaa 1020
ggagagaaaa ccagaagaac ccagccttta actggtttta tttatgtcat tgtcacaaat 1080
cctagtaggg gaaaaacaaa acaaagcttt tttaaaatca ctgaagctgc tgaaagaaaa 1140
gacacccttg tgcattggcc gggaatcgaa cccgggcctc ccgcgtggca ggcgagaatt 1200
ctaccactga accaccaatg catcttgtgg aacggctgtt tgcagcatga tctcttaaca 1260
tcatccctag ccctaggtcc cttcctagct gttcttctca gttataattt tgataatact 1320
gcctttatgt ttcaagaacc ccctcccctg caaatattaa ggattaagaa atttaaatct 1380
ttggccttga ggccatggct ctcttcccat ggtgacattt tctttgggct tttctggagg 1440
tgccggactc agacctgcct tcttcaaccc aggagggtgt tcttttgaga agttactgcc 1500
ctacctgact tctgccccac cgatggcctt tagaaccagc tgggagaagc tggaactttc 1560
tctgtggctc tgagtcagac gcaggtgagg ccacccttcc ttgaacagac gatccgcccg 1620
aaccttcttc ccgagtcagc ggctgagtca gccagagcgg gcgggctggg cggctctagg 1680
ttcgcccgga aggcgctgga aggttcacgg ttgcgggggc gggaggaggc gggtcttccg 1740
caactggata aaaagctcct ggaaggggag ggagagcaga tccgagctgg gtggacgcaa 1800
gagtggcagt gctgagcgta cctccaacgt tgagaagcac taaattccga aagctggtgc 1860
atctgacttc atattgcatt cgtggacaga agccacagcc atgtccgctg caagagaagt 1920
ggccataggc atcgacttgg gtaccacata ctcctgcgtg ggtgtgttcc agcacggtcg 1980
ggtggagatc ctagcaaatg accagggcaa ccgcaccacc cccagctacg tggccttcac 2040
cgacaccgag cggctggtgg gcgatgcagc caagagccag gcggccctga acccgcagaa 2100
taccgtgttt gacgccaagc ggctaattgg gcgcaagttt gcagacccca ccgtgcagtc 2160
agacttgaag cactggcctt tccaggtggt gagcgagggt gggaagccca aggtgcgcgt 2220
ttcctaccgt ggggaggaca aggcgttcta cccggaggag atttcgtcca tggtgctgag 2280
caagatgaag gagactgcgg aggcgtacct gggccagccc gtgaggcacg ctgtgatcac 2340
cgtgcctgcc tacttcaacg actctcagcg tcaggccacc aaggacgcag gtgccattgc 2400
agggctcaac gtgctgagga tcattaatga gcccacagct gctgccatcg cctatggcct 2460
ggaccggagg ggcgctggtg agcggaacgt gctcattttc gacttgggtg gaggaacctt 2520
cgacgtgtcg gtccttacta ttgacgcagg tgtctttgag gtgaaggcca cggctggaga 2580
cactcacctg ggtggagagg acttcgacaa ccgtttggtg aaccacttca tggaggagtt 2640
tcgacggaag cacaggaagg acttaagcac gaacaaacgg gccctgcgca ggctccgcac 2700
cgcctgtgag cgcgccaaac gcaccctgtc ttccagcacc caggccacgc tggaaatcga 2760
ctccctcttt gagggtgtag acttctacac ttctatcact cgagcccgct ttgaagaact 2820
gtgctcagac ctcttccgca gcaccctgga gccagtggag aaagccctgc gggatgccaa 2880
gctggacaag gctcagatcc atgatattgt cctagtgggt ggctctaccc gcatccccaa 2940
gatacagaag cttctgcagg acttcttcaa tggccgggaa ctgaacaaga gcataaaccc 3000
agatgaggct gtggcctatg gtgctgctgt gcaggcagca gtgttgatgg gggacaagtg 3060
tgagaaggtg caggatctcc tgctgctgga tgtggctccc ctgtccctgg ggctggagac 3120
agccgggggg gtgatgacca cgctgatcca gaggaatgcc accatcccta caaagcagac 3180
ccagactttc actacttatt cagacaacca acctggagtc ctgatccagg tgtatgaggg 3240
tgagagggcc atgaccaggg acaacaacct tctggggcgc tttgagctca gtggcatacc 3300
tcctgcccca cgtggagtgc cccagattga ggtgaccttc gacattgatg ccaatggcat 3360
cctgagtgtg acagccactg acaggagcac aggcagagct aacaagatca ctatcaccaa 3420
tgacaagggc cggctgagca aggaagaggt ggagaggatg gttcgtgagg cggagcagta 3480
caaggttgag gatgaggccc agagggaccg ggtggctgcc aaaaactccc tggaggccta 3540
tgttttccat gtgaagggct ccttgcacga ggaaagcctt agagacaaga ttcctgaaga 3600
ggacaggtgc aaagtacaag acaagtgtca ggaagtcctt acttggctgg agcacaacca 3660
gttggcagag aaagaggagt atgagcatca gaagagggaa ctggagcaga tttgtcgtcc 3720
catcttctcc aggctctatg ggggctctgg tatccctggg ggtagcagtt gtggtgctca 3780
agctcgacag ggagccccca gtactggccc tgtcattgag gaggttgatg ctactaactt 3840
cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatgg tgagcaaggg 3900
cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg 3960
ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct 4020
gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct 4080
gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt 4140
caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg 4200
caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga 4260
gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa 4320
ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa 4380
cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca 4440
gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca 4500
gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt 4560
gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtcctaa 4609
<210> 2
<211> 20
<212> DNA
<213> 人工序列()
<400> 2
cattgaggag gttgattaaa 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列()
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tttaatcaac ctcctcaatg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列()
<400> 4
taatcaacct cctcaatgac 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列()
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gtcattgagg aggttgatta 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列()
<400> 6
gtcacagctg acttgtcagg 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列()
<400> 7
cctgacaagt cagctgtgac 20
Claims (6)
1.一种能有效将应激信号转变为EGFP荧光信号的报告细胞系，其特征在于：其基因序列如SEQ no.1所示：
该报告细胞系能高效且快速地将HSPA6基因应激源对PK-15细胞的刺激信号转变为EGFP荧光信号。
2.如权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的制备方法，其特征在于所设计的sgRNA序列如下：
SgRNA-1序列：5- CATTGAGGAGGTTGATTAAA-3；
SgRNA-1作用位点的序列：5- TTTAATCAACCTCCTCAATG-3；
SgRNA-2序列：5- TAATCAACCTCCTCAATGAC-3；
SgRNA-2作用位点的序列：5- GTCATTGAGGAGGTTGATTA-3；
SgRNA-3序列：5- GTCACAGCTGACTTGTCAGG-3；
SgRNA-3作用位点的序列：5- CCTGACAAGTCAGCTGTGAC-3。
3.如权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的制备方法，包括以下步骤：
1）猪HSPA6基因特异sgRNA的设计和筛选
a. sgRNA的设计，根据猪HSPA6基因碳端的DNA序列，选择合适的PAM序列（NGG）用于基因编辑sgRNA靶点的设计， 所设计的sgRNA序列如权利要求2所示；
b.高效sgRNA的筛选，构建含上述sgRNA的表达载体，然后电转入细胞，通过测序等方法筛选出切割效率高且潜在脱靶效率低的sgRNA；
2）根据靶位点的序列，设计并合成EGFP同源打靶载体
根据上述筛选出的sgRNA，结合该sgRNA对HSPA6基因的的精确切割位点，设计设计并合成EGFP同源打靶载体；该打靶载体的主要原件依次为：上游同源臂、p2A位点、EGFP基因、下游同源臂及原核表达的骨架载体；
3）对权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的筛选和鉴定
a. sgRNA表达质粒和EGFP同源打靶载体的共转染；
b.极限稀释细胞铺板；
c. 细胞克隆的挑取和鉴定；
4）对权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系的荧光报告功能分析及验证；
a.HSPA6应激源对上述EGFP荧光报告细胞系的刺激实验；
b. EGFP荧光报告细胞系的报告能力评估。
4.如权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在制备HSPA6基因特异的活细胞传感器中的用途。
5.如权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在基因互作、启动子活性区域分析、病原检测、环境监测、食品安全、药物及毒物研究等方面的应用。
6.如权利要求1所述的能有效将应激信号转变为EGFP荧光信号的报告细胞系在制备HSAP6基因相同位点制备荧光报告动物模型中的应用。
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| CN113621610A (zh) * | 2021-06-21 | 2021-11-09 | 吉林大学重庆研究院 | 一对敲除猪HSPA6基因部分保守区域的sgRNA序列及应用 |
| CN115927298A (zh) * | 2021-10-05 | 2023-04-07 | 东北林业大学 | 马鹿miR PC-2869的前体及其应用 |
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| CN115927298A (zh) * | 2021-10-05 | 2023-04-07 | 东北林业大学 | 马鹿miR PC-2869的前体及其应用 |
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