CN106755026A - sgRNA表达载体的构建及牙釉质钙化不全模型的建立 - Google Patents
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Abstract
一种sgRNA表达载体的构建及牙釉质钙化不全模型的建立，属于生物技术领域。本发明的目的是采用CRISPR‑CAS9基因敲除技术成功获得人类牙釉质钙化不全模型的sgRNA表达载体的构建及牙釉质钙化不全模型的建立。本发明sgRNA表达载体的构建：sgRNA选取：在FAM83H基因第5个外显子处选取2个sgRNA序列作用靶点，合成两对寡聚核苷酸链，sgRNA双链DNA片段的合成，UC57和sgRNA双链DNA连接，使用sgRNA表达载体建立牙釉质钙化不全模型。本发明能更有效地预测新疫苗、新药和新诊断试剂等在临床应用中的效果，同时大大降低新药研发的风险，为临床研究提供基础模型。
Description
技术领域
本发明属于生物技术领域。
背景技术
牙釉质结构异常所导致的一组遗传性疾病，在不涉及全身系统疾病时称之为遗传性牙釉质发育不全（amelogenesis imperfecta，AI）。根据临床表现分为三型，牙釉质钙化不全较为常见。目前已明确某些基因的突变是导致AI 的根本原因，其中Fam83h（Familywith sequence similarity 83， member H，Fam83h）基因突变导致的AI 占有重要比例。临床上有大量的AI 患者，牙釉质发育不全严重影响着患者的美观、咬合关系、生活等方面。
人类疾病的病症模型是疾病机理研究和新药研发的重要基础。因此建立人类疾病模型，有效地模拟人类疾病的病理过程，能更有效地预测新药和新诊断试剂等在临床应用中的效果，同时大大降低新药研发的风险。
发明内容
本发明的目的是采用CRISPR-CAS9基因敲除技术成功获得人类牙釉质钙化不全模型的sgRNA表达载体的构建及牙釉质钙化不全模型的建立。
本发明sgRNA表达载体的构建：
①sgRNA选取：在FAM83H基因第5个外显子处选取2个sgRNA序列作用靶点，合成两对
寡聚核苷酸链
（sgRNA1: F:TAGGCTGGGCGAACGAGTCGCGC
和R:AAACGCGCGACTCGTTCGCCCAG；
sgRNA2:F:TAGGCTTCGAGGTGTTCTGCAAG
和R:AAACCTTGCAGAACACCTCGAAG
）用于制备sgRNA；该sgRNA的寡聚核苷酸链选取原则：选取分数最高并碱基序列开头为GG的寡聚核苷酸链；
②sgRNA双链DNA片段的合成：将合成的寡聚核苷酸链按照sgRNA双链DNA合成的反应体系混合，并经过95℃变性5min，室温放置30min，以便形成双链DNA片段；sgRNA双链DNA合成的反应体系：
；
③PUC57载体线性化：PUC57克隆载体按照酶切体系经BbsⅠ酶切线性化
酶切体系：质粒PUC57 20μl
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
酶切37℃ 3h，电泳跑胶后，使用DNA琼脂糖胶回收试剂盒进行回收；
④PUC57和sgRNA双链DNA连接：按照sgRNA双链DNA连接反应体系将PUC57和双链DNA连接，16℃连接过夜；sgDNA连接反应体系：
。
本发明sgRNA表达载体核苷酸序列是SEQ ID NO：1、SEQ ID NO：2。
本发明使用sgRNA表达载体建立牙釉质钙化不全模型：
①转化：
a、从-80℃冰箱取50 μL感受态细菌，加入10 μL连接产物，混匀，放于冰上30分钟；
b、42℃水浴热击90s，放于冰上2分钟；
c、加200μL LB液体培养基，在恒温震荡培养箱37℃，250 rpm震荡培养30 min；
d、吸取200 μL菌液均匀地涂布在氨苄抗性LB平板上，放于37℃恒温培养箱中培养12小时；
②单克隆的挑取：从培养基中挑取单菌落，接种于6 mL液体LB培养基中（含6 μL氨苄青霉素），放于摇床中，37℃培养12-14h；
③质粒DNA的提取：将细菌中的质粒提取出来；
④质粒测序鉴定：使用M13通用引物对质粒进行测序分析，测序连接正确后备用；
⑤受精卵显微注射：将CAS9mRNA/sgRNA混合并注射到细胞质中，其中CAS9mRNA终浓度为150ng/μl，sgRNA终浓度为30ng/μl；
⑥注射后的受精卵移植及动物培育：显微注射后，将受精卵移植，进行胚胎发育，进行规范化饲养。
本发明经过相关检测，成功获得牙釉质钙化不全模型，该模型的获得能够有效地模拟人类疾病的病理过程，能更有效地预测新疫苗、新药和新诊断试剂等在临床应用中的效果，同时大大降低新药研发的风险，为临床研究提供基础模型。
附图说明
图1是本发明表达载体PUC57-sgRNA的结构示意图；
图2是本发明PCR产物鉴定胚胎FAM83H基因敲除情况的电泳图；其中Mark D2000 为DNA分子标准量；1为水对照；2为阴性对照（正常胚胎）；3为空泳道；4-13:显微注射后10个胚胎DNA PCR结果。设计的FAM83H基因的鉴定引物为1224bp，从DNA测序结果和PCR产物电泳结果可以得到：4，5，6，7，8，9，10，11，13均发生不同的敲除情况；12没有发生敲除；
图3是显微注射后得到的新生个体经鉴定后，将对照组和敲除组分别拍照记录牙齿外观异样情况；该图为a为正常组、b为单敲组、c为双敲组新生个体的外观照，从图上可以看出单敲组和双敲组牙齿的颜色出现发黄现象；
图4是显微注射后得到的个体经鉴定后，将正常组和敲除组分别进行牙齿组织切片的结果：
该图为正常组合敲除组个体牙齿的变化，可以看出敲除组个体牙齿发生牙骨质稀疏。
具体实施方式
本发明sgRNA表达载体的构建：
①sgRNA选取：在FAM83H基因第5个外显子处选取2个sgRNA序列作用靶点，合成两对
寡聚核苷酸链
（sgRNA1: F:TAGGCTGGGCGAACGAGTCGCGC
和R:AAACGCGCGACTCGTTCGCCCAG；
sgRNA2:F:TAGGCTTCGAGGTGTTCTGCAAG
和R:AAACCTTGCAGAACACCTCGAAG
）用于制备sgRNA；该sgRNA的寡聚核苷酸链选取原则：选取分数最高并碱基序列开头为GG的寡聚核苷酸链。
②sgRNA双链DNA片段的合成：将合成的寡聚核苷酸链按照sgRNA双链DNA合成的反应体系混合，并经过95℃变性5min，室温放置30min，以便形成双链DNA片段；sgRNA双链DNA合成的反应体系：
。
③PUC57载体线性化：PUC57克隆载体按照酶切体系经BbsⅠ酶切线性化
酶切体系：质粒PUC57 20μl
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
酶切37℃ 3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒购于天根公司，北京，中国）进行回收；具体操作按说明书进行。
④PUC57和sgRNA双链DNA连接：按照sgRNA双链DNA连接反应体系将PUC57和双链DNA连接，16℃连接过夜；sgDNA连接反应体系：
。
本发明sgRNA表达载体核苷酸序列是SEQ ID NO：1、SEQ ID NO：2。
本发明使用sgRNA表达载体建立牙釉质钙化不全模型：
①转化：
a、从-80℃冰箱取50 μL感受态细菌，加入10 μL连接产物，混匀，放于冰上30分钟；
b、42℃水浴热击90s，放于冰上2分钟；
c、加200μL LB液体培养基，在恒温震荡培养箱37℃，250 rpm震荡培养30 min；
d、吸取200 μL菌液均匀地涂布在氨苄抗性LB平板上，放于37℃恒温培养箱中培养12小时。
②单克隆的挑取：从培养基中挑取单菌落，接种于6 mL液体LB培养基中（含6 μL氨苄青霉素），放于摇床中，37℃培养12-14h。
③质粒DNA的提取：将细菌中的质粒提取出来。
④质粒测序鉴定：使用M13通用引物对质粒进行测序分析，测序连接正确后备用；可用于后续实验。
⑤受精卵的获取和显微注射：注射卵泡刺激素（FSH），之后注射人绒毛膜促性腺激素(HCG)（购于宁波第二激素厂），获取受精卵，通过显微注射仪器将预混好CAS9mRNA/sgRNA混合物注射到细胞质中 (CAS9mRNA终浓度为150ng/μl，sgRNA终浓度为30ng/μl）。
CAS9表达质粒（Addgene，实验室购买），经酶切线性化，经酚氯仿抽提纯化后，溶于无核酸酶的水中作为模板，用于体外转录。CAS9mRNA的合成由试剂盒RNeasy Mini Kit(Qiagen,No.74104)在体外作用T7RNA聚合酶来完成，sgRNA的体外合成由试剂盒MiRNeasyMini Kit(Qiasgen,No.217004)在体外利用T7RNA聚合酶完成。
酶切体系： NotⅠ 4μl
CAS9 50μl
BSA 30μl
Triton 30μl
10×H 30μl
ddH2O 156μl
酶切37℃ 3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒（购于天根公司，北京，中国）进行回收，具体操作按说明书进行。
⑥注射后的受精卵移植及动物培育：显微注射后，将受精卵移植，进行胚胎发育，进行规范化饲养。
敲除验证：
受精卵的体外培养和发育：将显微注射的受精卵转移到培养液中，置于37℃恒温培养箱中培养，发育到桑椹胚时期时，用吸卵针将单个胚胎转移到离心管中，用于后面实验。
胚胎FAM83H基因敲除情况鉴定：
显微注射后的胚胎，体外培养5d后，取出发育至桑椹胚期的胚胎，放于PBS中清洗3次后，收集单个胚胎至于PCR管中。在单个胚胎中加入5μL NP40裂解液进行胚胎裂解。裂解条件为：56℃，1 h；95℃，10 min。以裂解产物为模板，使用PCR上游和下游引物进行PCR扩增，电泳鉴定，并进行DNA测序，得到基因型鉴定结果。
设计PCR引物如下：
上游引物：CACAGCAAGGCTGTCGTGTCC
下游引物：GAACTTGCCCACCTTGCTGTC。
PCR反应体系如下：
模板DNA 1ul
上游引物 1ul
下游引物 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul。
PCR反应条件：
95℃预变性5min；94 ℃变性30s，58℃退火30 s，72 ℃延伸40s；35个循环；72 ℃延伸5min。
PCR产物进行测序，若测序结果在FAM83H基因引物设计的打靶位点附近出现双峰的情况,则为打靶成功。选择双峰的样品再次PCR，产物胶回收后进行连接PGM-T载体，转化后挑取阳性克隆再次进行测序，测序结果中在FAM83H基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变,则判断为基因敲除。
牙釉质钙化不全模型表型鉴定和基因型分析：
1）表型结果统计：出生后1周分别对敲除和正常个体进行牙齿外观照片采集。
2）DNA测序鉴定FAM83H基因敲除的牙釉质钙化不全模型的基因型：提取组织DNA，提取方法按照组织基因组提取试剂盒说明书进行操作（天根，北京，中国），进行PCR，电泳鉴定，并进行DNA测序，得到基因型鉴定结果。
设计PCR引物如下：
上游引物：CACAGCAAGGCTGTCGTGTCC
下游引物：GAACTTGCCCACCTTGCTGTC
PCR反应体系如下：
模板DNA 1ul
上游引物 1ul
下游引物 1ul
2×Taq plus 12.5ul
ddH2O 9.5ul
PCR反应条件：
95℃预变性5min；94 ℃变性30s，58℃退火30 s，72 ℃延伸40s；35个循环；72 ℃延伸5min。
PCR产物进行测序，若测序结果在FAM83H基因引物设计的打靶位点附近出现双峰的情况,则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后进行连接T载体，转化后挑取阳性克隆再次进行测序，若测序结果中在FAM83H基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变,则可判断为基因敲除。
Western Blot 具体步骤如下：
样品准备，首先取10ug蛋白样品，加上上样缓冲液，沸水变性5分钟，立即放到冰上，12000rpm，4℃离心5分钟。
电泳：将蛋白样品上样到已配好的12%聚丙烯胺凝胶的样品孔内，进行电泳，电泳至溴酚蓝刚跑出既可终止电泳，进行转膜。
转膜：1） 准备一张 6 层的滤纸和一张 PVDF 膜。将切好的 PVDF 膜置于甲醇中浸泡至少5 分钟后使用。
2）准备好两个培养皿，一个加入转膜液，一个放甲醇。
3）将夹子打开使黑的一面在下。在上面垫一张海绵垫，用玻璃棒来回擀几遍以擀走海绵垫里的气泡。另一手压住海绵垫使其不能随便移动。在垫子上铺三层滤纸，要保证三层滤纸叠放的整齐，一手固定滤纸一手用玻棒擀去其中的气泡。从架子上取下玻璃板，然后轻轻将玻璃板撬开。除去一块玻璃板后，将浓缩胶轻轻切去，避免将分离胶扯断。小心切去需要部位的分离胶，然后将其放到滤纸上，并将它和滤纸对齐，注意不要产生气泡。将已浸过甲醇的PVDF 膜盖于胶上，要盖满整个胶，注意胶和膜之间一定不能有气泡。然后在膜上轻轻盖上 3张滤纸。最后盖上海绵垫，合起夹子，整个操作需在转膜液中进行。
4）将夹子放入转移槽中，要使夹子黑面对着槽的黑面，夹子的白面对着槽的红面。电转移时会产热，槽需用冰块降温。用 100V 转膜2小时。
洗膜：取出膜，放入TBST中洗15分钟，连续洗3次。
封闭：将膜于5%脱脂乳的TBST溶液中封闭2小时。
一抗孵育：用含有5%脱脂乳的TBST进行一抗稀释，4℃孵育过夜。
洗膜：将膜用TBST洗15分钟，连续3次。
二抗:用含有5%脱脂乳的TBST进行二抗稀释，孵育2小时。
洗膜：将膜用TBST洗15分钟，连续3次。
显影：需要配显影液，使用显影仪进行显影。
（3）新生个体牙釉质钙化不全模型表型鉴定和基因型分析。
1）新生个体牙齿外观变化采集。
出生后1周分别对敲除组和正常组进行个体牙齿外观照片采集。
2）观察新生个体牙齿组织是否发生病变。
敲除个体在生长过程中，出现死亡的个体，固定牙齿组织，对牙齿进行脱钙处理，做组织病理切片。
<110> 吉林大学
<120> 采用敲除技术建立牙釉质钙化不全模型的方法
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1000
<212> DNA
<213> 人工序列
<400> 1
gccagctgta ttggagatcg gtacttcgcg aatgcgtcga gatattgggt ctttaaaagc 60
accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt 120
tctagctcta aaacgcgcga ctcgttcgcc cagcctatag tgagtcgtat taattgggta 180
tcggatgccg ggaccgacga gtgcagaggc gtgcaagcga gcttggcgta atcatggtca 240
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 300
agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 360
cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 420
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 480
tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 540
cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 600
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 660
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 720
agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 780
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 840
cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 900
ccccccgttc agcccgaccg ctgcgcctta tcccggtaac tatcgtcttg agtccaaccc 960
gggtaagaca cgacttatcg ccactggcag cagccactgg 1000
<160> 2
<170> PatentIn version 3.5
<210> 2
<211> 1000
<212> DNA
<213> 人工序列
<400> 1
attggggaga tcggtacttc gcgaatgcgt cgagatattg ggtctttaaa agcaccgact 60
cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct atttctagct 120
ctaaaacctt gcagaacacc tcgaagccta tagtgagtcg tattaattgg gtatcggatg 180
ccgggaccga cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt 240
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 300
agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 360
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 420
cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 480
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 540
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 600
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 660
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 720
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 780
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 840
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 900
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 960
acgacttatc gccactggca gcagccactg gtaacaggat 1000
Claims (3)
1.一种sgRNA表达载体的构建，其特征在于：
①sgRNA选取：在FAM83H基因第5个外显子处选取2个sgRNA序列作用靶点，合成两对
寡聚核苷酸链
（sgRNA1: F:TAGGCTGGGCGAACGAGTCGCGC
和R:AAACGCGCGACTCGTTCGCCCAG；
sgRNA2:F:TAGGCTTCGAGGTGTTCTGCAAG
和R:AAACCTTGCAGAACACCTCGAAG
）用于制备sgRNA；该sgRNA的寡聚核苷酸链选取原则：选取分数最高并碱基序列开头为GG的寡聚核苷酸链；
②sgRNA双链DNA片段的合成：将合成的寡聚核苷酸链按照sgRNA双链DNA合成的反应体系混合，并经过95℃变性5min，室温放置30min，以便形成双链DNA片段；sgRNA双链DNA合成的反应体系：
；
③PUC57载体线性化：PUC57克隆载体按照酶切体系经BbsⅠ酶切线性化
酶切体系：质粒PUC57 20μl
10×buffer 20μl
BbsⅠ 1μl
ddH2O 159μl
酶切37℃ 3h，电泳跑胶后，使用DNA琼脂糖胶回收试剂盒进行回收；
④PUC57和sgRNA双链DNA连接：按照sgRNA双链DNA连接反应体系将PUC57和双链DNA连接，16℃连接过夜；sgDNA连接反应体系：
。
2.根据权利要求1所述的sgRNA表达载体的构建，其特征在于：sgRNA表达载体核苷酸序列是SEQ ID NO：1、SEQ ID NO：2。
3.使用sgRNA表达载体建立牙釉质钙化不全模型，其特征在于：
①转化：
a、从-80℃冰箱取50 μL感受态细菌，加入10 μL连接产物，混匀，放于冰上30分钟；
b、42℃水浴热击90s，放于冰上2分钟；
c、加200μL LB液体培养基，在恒温震荡培养箱37℃，250 rpm震荡培养30 min；
d、吸取200 μL菌液均匀地涂布在氨苄抗性LB平板上，放于37℃恒温培养箱中培养12小时；
②单克隆的挑取：从培养基中挑取单菌落，接种于6 mL液体LB培养基中（含6 μL氨苄青霉素），放于摇床中，37℃培养12-14h；
③质粒DNA的提取：将细菌中的质粒提取出来；
④质粒测序鉴定：使用M13通用引物对质粒进行测序分析，测序连接正确后备用；
⑤受精卵显微注射：将CAS9mRNA/sgRNA混合并注射到细胞质中，其中CAS9mRNA终浓度为150ng/μl，sgRNA终浓度为30ng/μl；
⑥注射后的受精卵移植及动物培育：显微注射后，将受精卵移植，进行胚胎发育，进行规范化饲养。
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