CN112695081A - 原发性胆汁性胆管炎新的易感基因及其应用 - Google Patents
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Abstract
本发明提供一种原发性胆汁性胆管炎的新突变SNP位点,其特征在于,所述新突变SNP位点为人PTK2B基因外显子24,NM_173174.2,c.1679C→G,p.560,Pro(P)→Arg(R)。本发明发现一个原发性胆汁性胆管炎患者新的易感基因,为疾病诊断新增了一个新的标志物,由于该病早期常无明显症状,且现有抗体检测敏感性有限,在部分患者中仍呈阴性,待患者血清学指标(碱性磷酸酶、γ‑谷氨酰转肽酶、胆红素)有明显升高时往往已进入疾病中晚期,对药物反应不佳,严重影响预后,因此新的标志物有助于实现疾病早期诊断,以早期治疗,从而改善患者预后。
Description
技术领域
本发明属于生物医学技术领域,具体涉及一种原发性胆汁性胆管炎新的易感基因及其应用。
背景技术
原发性胆汁性胆管炎(Primary biliary cholangitis,PBC),又称原发性胆汁性肝硬化(Primary biliary cirrhosis,PBC),是好发于中年女性的一种慢性自身免疫性疾病,患病率在世界不同地区为20-40/10万,中国患病率数据有限,中国南方为49.2/10万。
PBC是主要以肝内小胆管的慢性非化脓性炎症为特征的自身免疫病,临床主要为胆汁淤积和肝功能减退的表现如黄疸、瘙痒、乏力等,血清学有碱性磷酸酶(Alkalinephosphatase,ALP)、γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,GGT)升高,多有抗线粒体抗体(Anti-mitochondrial antibody,AMA)阳性。病理表现为肝内小胆管非化脓性肉芽肿性慢性炎症。
该病目前病因不明,较公认的观点是遗传因素及环境因素共同作用所致。有研究发现,PBC患者一级亲属PBC发生率高于正常人群约100倍,姐妹患病的相对风险为10左右,同卵双胞胎中同病一致率为63%,且发病年龄相似,而在异卵双生胎中没有共同发病。以上,均提示遗传因素在PBC中起重要作用。近年来随着第二代DNA测序技术(Nextgeneration sequencing,NGS)的发展,加速和改变了对遗传性疾病的研究模式,提高了疾病研究效率。在不同种族的PBC人群中筛查到了不同的易感基因位点,如欧洲PBC患者中的易感基因为IL12A,IL12RB2,CD80,STAT4,CXCR5等,日本PBC患者中的易感基因为TNFSF15和POU2AF1,而在中国汉族PBC人群中,通过对14个SNPs检测显示存在易感基因TNFSF15和CD80,但并未发现POU2AF1和IL12A等易感基因,提示中国汉族PBC患者与欧洲、日本人群存在某些共同的易感基因,但同时又有自己不同的遗传易感性特征。
全外显子测序(Whole exome sequencing,WES)技术是以人类基因组中蛋白质编码序列为目的序列的第二代测序技术,外显子组(Exome)仅占不到人类整个基因组的1%,但在已知的人类单基因遗传病中,位于外显子区域的致病突变基因占绝大多数,该技术快速、高效、性价比高,被Science杂志评为2010年十大科学突破之一。散发PBC人群的NGS结果均显示PBC存在遗传易感性,而针对PBC的家族聚集现象,目前国际上尚无通过全外显子测序筛查中国汉族PBC家系易感基因的研究,因此,拟针对PBC患者家系进行全外显子测序,以筛查易感基因,更好的阐释该病的遗传学特征。
现有研究认为原发性胆汁性胆管炎是一种由于遗传因素和环境因素共同作用导致的疾病,而该病的一级亲属发病率远远高于普通人群,提示遗传因素在发病中起着重要作用。在现有的全基因组关联分析研究中,报道了原发性胆汁性胆管炎散发患者的相关易感基因,但目前尚没有关于中国汉族人群原发性胆汁性胆管炎患者家系易感基因的报道,且前期全基因组关联分析研究报道的相关易感基因后续暂未有关于基因功能进一步的动物实验。此外,目前该病尚没有非常好的动物模型。
发明内容
为解决上述问题,本发明提供一种原发性胆汁性胆管炎的新突变SNP位点。
本发明提供的一种原发性胆汁性胆管炎的新突变SNP位点为人PTK2B基因外显子24,NM_173174.2,c.1679C→G,p.560,Pro(P)→Arg(R)。
本发明还提供一种用于鉴定所述的原发性胆汁性胆管炎的新突变SNP位点的引物对,引物序列如下:
引物-F:ACAGTGTAGGCAACAGCACAGAAG(SEQ ID No.2)
引物-R:CGTGAAGCGTCGGAAGTTAATGGA(SEQ ID No.3)
本发明还提供一种用于鉴定所述的原发性胆汁性胆管炎的新突变SNP位点的试剂盒,其含有所述的引物对。任选地,所述试剂盒还可包括dNTPs、Taq酶、Mg2+和/或PCR反应缓冲液。
本发明还提供所述的新突变SNP位点在制备原发性胆汁性胆管炎检测、诊断、治疗和/或预后产品中的应用。
其中,所述产品为试剂盒,所述试剂盒包括用于扩增基因PTK2B外显子24新突变SNP位点的引物对。
其中,所述引物对的核苷酸序列如SEQ ID No.2-3所示。任选地,所述试剂盒还可包括dNTPs、Taq酶、Mg2+和/或PCR反应缓冲液。
PCR反应体系(25μl):
PCR循环反应条件:98℃变性2分钟;98℃10秒,60℃30秒,72℃30秒,共35个循环;72℃延伸7分钟。
对于疑诊原发性胆汁性胆管炎的患者,但抗线粒体抗体(AMA)、抗sp100、抗gp210等抗体检测呈阴性无法明确诊断,可通过抽取外周血2-4ml进行该基因突变位点检测,若检测到C→G点突变,则为疾病诊断更进一步增加了证据。检测的引物和PCR反应体系和程序如前所述。
本发明还提供一种原发性胆汁性胆管炎的动物模型的构建方法,其包括如下步骤:
1)设计PTK2b基因19号外显子靶向的sgRNA识别序列,构建sgRNA;
2)设计构建有PTK2B-P560R片段的供体质粒;
3)将Cas9 mRNA,sgRNA和供体共注射到小鼠受精卵,受精卵在交配后0.5天被移植到ICR雌性小鼠的输卵管中,移植后的19-21天F0代小鼠出生;
4)将携带有PTK2B基因点突变的F0代小鼠和C57BL/6J回交以得到杂合突变小鼠;
5)将携带有PTK2B基因点突变的F1代雌雄小鼠进行繁育,得到携带基因突变的后代小鼠。
其中,sgRNA的序列如SEQ ID No.4-5所示。
本发明发现一个原发性胆汁性胆管炎患者新的易感基因,为疾病诊断新增了一个新的标志物,由于该病早期常无明显症状,待患者血清学指标(碱性磷酸酶、γ-谷氨酰转肽酶、胆红素)有明显升高时往往已进入疾病中晚期,严重影响预后,因此新的标志物有助于实现疾病早期诊断,以早期治疗,从而改善患者预后。
本发明成功建立了携带基因突变的小鼠繁育体系,携带基因突变的小鼠出现了原发性胆汁性胆管炎疾病的表型,更加证实了突变基因在发病机制中的作用。并且利用该基因构建的小鼠解决了现有关于原发性胆汁性胆管炎动物模型不够理想的现状,从而为后续疾病研究提供了有效的动物模型,解决了由于患者肝脏穿刺为有创操作、存在风险而不易获取患者肝脏组织标本的困难,有利于更好的开展疾病深入研究。
附图说明
图1为本发明专利在家系中发现的新基因示意图;图中,A,原发性胆汁性胆管炎患者家系图;B,家系内发现的原发性胆汁性胆管炎患者存在的突变位点;C,与突变位点对应的正常序列图。
图2所示为含有PTK2B-P560R片段的供体质粒示意图。
图3所示为电泳图。B6为阴性对照,是B6基因组DNA;N为空白对照,无模板的对照;DL2000条带:2000bp,1000bp,750bp,500bp,250bp,100bp;P:阳性质粒对照;Trans2K条带:8000bp,5000bp,3000bp,2000bp,1000bp,750bp,500bp,250bp,100bp。A,利用ZMK2F4及3004Ptk2b-KITR1引物进行PCR产物的电泳图,提示29、37、38、39号F1代小鼠携带突变;B,利用3004ptk2b-wtTF及3004ptk2b-wtTR引物进行PCR产物的电泳图,提示29、37、38、39号F1代小鼠为杂合突变;C及D,29、37、38、39号F1代小鼠分别利用3004ptk2b-5InF1、3004ptk2b-5InR1引物及ZMK2F4、3004ptk2b-3InR2引物进行PCR的产物的电泳图,以进行后续测序;E,利用ZMK2F4及3004Ptk2b-KITR1引物进行PCR产物的电泳图,提示40、41、43号F1代小鼠携带突变;F,利用3004ptk2b-wtTF及3004ptk2b-wtTR引物进行PCR产物的电泳图,提示40、41、43号F1代小鼠为杂合突变;G,40、41、43号F1代小鼠分别利用3004ptk2b-5InF1、3004ptk2b-5InR1引物及ZMK2F4、3004ptk2b-3InR2引物进行PCR的产物的电泳图,以进行后续测序。
图4所示为小鼠基因型Sanger测序结果。
图5为将该基因敲入小鼠后小鼠表型示意图。图中,A,携带基因突变的雌性10月龄小鼠肝脏病理;B,携带基因突变的雌性8月龄小鼠肝脏病理;C,携带基因突变的雌性3月龄小鼠血清抗线粒体抗体结果。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1原发性胆汁性胆管炎新的易感基因筛选
家系内所有成员采集外周血,提取外周血DNA,家系内3个患者(I-2,I-3,I-4)和1个健康对照者(I-6)基因组DNA进行全外显子测序,包括:
1.文库制备:1)基因组片段化:Cavoris仪打断至200bp左右。2)末端补平修复:片段化DNA在Klenow Fragment,T4 DNA polymerase和T4PNK的作用下进行末端补平修复。3)3’端腺苷化:在聚合酶体系下,使上一步得到的修复产物在3’末端加上A碱基,为下一步连接做好准备。4)加接头:配置T4DNA连接酶反应体系,在Thermo mixer中适温反应一定时间使adapter和加“A”产物连接。5)扩增:连接产物经4-6轮LM-PCR扩增。6)杂交:文库与探针混于杂交体系中65℃,60-68h杂交。7)洗涤磁珠和洗脱DNA:使用链霉素磁珠,与杂交样本孵育后洗脱液洗脱。8)洗脱产物扩增:洗脱产物经10轮LM-PCR扩增。
2.Illumina平台测序:1)测序,Illumina hiseq2500平台标准化上机测序操作流程。2)测序得到图像原始数据,Illumina官方basecall分析软件BclToFastq得到原始数据(raw data)。3)数据分析:去接头污染,去低质量的数据;数据与参考序列比对统计(比对软件BWA),参考基因组为hg19基因组;SNP检测及注释:分析软件samtools;Indel检测及注释:分析软件pindel;突变假阳性过滤:根据测序深度,突变质量,对检测得到的SNP,Indel进行过滤筛选,得到高质量可靠的突变;突变注释:SNP和Indel根据在基因上的位置,分析得到氨基酸变化影响,剪切影响,UTR,内含子突变影响等;筛选出的变异对蛋白功能影响的预测:使用SIFT利用基于同源比对,蛋白结构的保守性等的算法,预测筛选出的变异对蛋白质的影响;对剪切位点附近的突变,做剪切危害性预测。SOAPaligner(soap2.21)能够对彻底读出的序列输出进行有效对比,比对参照序列为人类参考基因组hg19。
基于SOAP的对比结果,再使用SOAPsnp软件进行SNP分析,获得每个样本中外显子组SNP数据及相应SNP的基因分型。SOAPsnp不仅对SNP进行识别,还可以注释和重新校准SNP。比对出的SNP结果进行第一步的过滤,过滤挑选出SNP的标准为:1)基础质量数值超过20;2)测序深度在4-200之间;3)计算获得的SNP拷贝数等于或小于2;4)相邻SNP的距离不小于5bp。有关SOAPaligner和SOAPsnp的计算参数的可以在以下网址获得(http://soap.genomics.org.cn/)。经过一步过滤的SNP数据再过滤除去公共数据中已记录的SNP。过滤的公共数据库包括dbSNP、1000Genome Project和HAPmap。dbSNP、1000Genome Project公共数据中SNP相关数据可以通过NCBI下载(http://www.ncbi.nlm.nih.gov)。SNP对应相应蛋白质功能的影响可通过SIFT软件来预测。在经过SNP分析后,对所研究家系中的样本的全外显子组数据进行indel变异分析。
分别得到较为可靠的突变结果(突变深度至少2x,突变率至少10%以上)36022、36604、36265、37737个。筛选出I-2,I-3,I-4共有,而I-6未携带的突变146个。去掉已有rs号(已报告的SNPs)的突变,MAF选择0,千人基因组频率(中国),千人基因组频率(中国南方),千人基因组频率(中国北方)选择0,筛选出突变24个。突变类型去掉非编码区突变(保留错义、剪切位点、缺失移码),筛选出可能与PBC疾病发病相关的候选突变17个。将这17个突变位点通过Sanger测序在家系内非患者成员中进行验证,得到家系内共分离的基因PTK2B外显子24,NM_173174.2,c.1679C→G,p.560,Pro(P)→Arg(R),错义突变,序列如SEQ ID No.1所示。
实施例2
将PTK2B的基因突变位点利用Cas9/RNA系统基因定位敲入小鼠,具体制作流程:
1.sgRNA设计及构建:设计PTK2b基因19号外显子靶向的sgRNA识别序列,构建sgRNA;
表1 sgRNA序列
sgRNA名称 | sgRNA序列(5’→3’) | 荧光探针(PAM) |
PTK2B-S1 | ctcggggctcatgagctcaa | GGG |
PTK2B-S2 | tgctgtccggaacatcctgg | TGG |
2.供体设计及构建:设计构建有PTK2B-P560R片段的供体质粒,质粒示意图如图2所示。中靶载体序列如SEQ ID No.6所示,其中2159位的C突变成G。
3.将Cas9 mRNA,sgRNA和供体共注射到受精卵。之后受精卵在交配后0.5天被移植到ICR雌性小鼠的输卵管中,移植后的19-21天F0代小鼠出生。
4.F0代小鼠鉴定及繁育:通过取鼠尾DNA进行PCR和测序验证携带有PTK2B基因点突变的小鼠;将阳性F0代小鼠和C57BL/6J回交以得到杂合突变小鼠;
表2 PCR引物信息
表3测序引物信息
表3 F0代小鼠基因型检测
编号 | 性别 | 基因型 | 代数 |
1 | 雄 | + | F0 |
2 | 雄 | + | F0 |
4 | 雌 | + | F0 |
10 | 雄 | + | F0 |
13 | 雄 | + | F0 |
14 | 雌 | + | F0 |
5.F1代小鼠鉴定:通过PCR和测序验证阳性小鼠。将F1代雌雄小鼠进行繁育,得到携带基因突变的后代小鼠。
表4 F1代小鼠基因型检测
编号 | 性别 | 基因型 | 代数 |
29 | 雌 | 杂合突变 | F1 |
37 | 雄 | 杂合突变 | F1 |
38 | 雄 | 杂合突变 | F1 |
39 | 雌 | 杂合突变 | F1 |
40 | 雄 | 杂合突变 | F1 |
41 | 雄 | 杂合突变 | F1 |
43 | 雌 | 杂合突变 | F1 |
通过对肝脏病理及血清学进行观察检测,发现携带基因突变的小鼠肝脏病理符合原发性胆汁性胆管炎的病理表现(图5A),主要包括门脉炎症、胆管破坏、肝小叶炎症、肉芽肿形成等(图5B),血清学有抗线粒体抗体阳性(图5C),出现了该病的表型。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院北京协和医院
<120> 原发性胆汁性胆管炎新的易感基因及其应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3912
<212> DNA
<213> Homo sapiens
<400> 1
atgtctgggg tgtccgagcc cctgagtcga gtaaagttgg gcacgttacg ccggcctgaa 60
ggccctgcag agcccatggt ggtggtacca gtagatgtgg aaaaggagga cgtgcgtatc 120
ctcaaggtct gcttctatag caacagcttc aatcctggga aaaacttcaa actggtcaaa 180
tgcactgtcc agacggagat ccgggagatc atcacctcca tcctgctgag cgggcggatc 240
gggcccaaca tccggttggc tgagtgctat gggctgaggc tgaagcacat gaagtccgat 300
gagatccact ggctgcaccc acagatgacg gtgggtgagg tgcaggacaa gtatgagtgt 360
ctgcacgtgg aagccgagtg gaggtatgac cttcaaatcc gctacttgcc agaagacttc 420
atggagagcc tgaaggagga caggaccacg ctgctctatt tttaccaaca gctccggaac 480
gactacatgc agcgctacgc cagcaaggtc agcgagggca tggccctgca gctgggctgc 540
ctggagctca ggcggttctt caaggatatg ccccacaatg cacttgacaa gaagtccaac 600
ttcgagctcc tagaaaagga agtggggctg gacttgtttt tcccaaagca gatgcaggag 660
aacttaaagc ccaaacagtt ccggaagatg atccagcaga ccttccagca gtacgcctcg 720
ctcagggagg aggagtgcgt catgaagttc ttcaacactc tcgccggctt cgccaacatc 780
gaccaggaga cctaccgctg tgaactcatt caaggatgga acattactgt ggacctggtc 840
attggcccta aagggatccg ccagctgact agtcaggacg caaagcccac ctgcctggcc 900
gagttcaagc agatcaggtc catcaggtgc ctcccgctgg aggagggcca ggcagtactt 960
cagctgggca ttgaaggtgc cccccaggcc ttgtccatca aaacctcatc cctagcagag 1020
gctgagaaca tggctgacct catagacggc tactgccggc tgcagggtga gcaccaaggc 1080
tctctcatca tccatcctag gaaagatggt gagaagcgga acagcctgcc ccagatcccc 1140
atgctaaacc tggaggcccg gcggtcccac ctctcagaga gctgcagcat agagtcagac 1200
atctacgcag agattcccga cgaaaccctg cgaaggcccg gaggtccaca gtatggcatt 1260
gcccgtgaag atgtggtcct gaatcgtatt cttggggaag gcttttttgg ggaggtctat 1320
gaaggtgtct acacaaatca caaaggggag aaaatcaatg tagctgtcaa gacctgcaag 1380
aaagactgca ctctggacaa caaggagaag ttcatgagcg aggcagtgat catgaagaac 1440
ctcgaccacc cgcacatcgt gaagctgatc ggcatcattg aagaggagcc cacctggatc 1500
atcatggaat tgtatcccta tggggagctg ggccactacc tggagcggaa caagaactcc 1560
ctgaaggtgc tcaccctcgt gctgtactca ctgcagatat gcaaagccat ggcctacctg 1620
gagagcatca actgcgtgca cagggacatt gctgtccgga acatcctggt ggcctcccgt 1680
gagtgtgtga agctggggga ctttggtctt tcccggtaca ttgaggacga ggactattac 1740
aaagcctctg tgactcgtct ccccatcaaa tggatgtccc cagagtccat taacttccga 1800
cgcttcacga cagccagtga cgtctggatg ttcgccgtgt gcatgtggga gatcctgagc 1860
tttgggaagc agcccttctt ctggctggag aacaaggatg tcatcggggt gctggagaaa 1920
ggagaccggc tgcccaagcc tgatctctgt ccaccggtcc tttataccct catgacccgc 1980
tgctgggact acgaccccag tgaccggccc cgcttcaccg agctggtgtg cagcctcagt 2040
gacgtttatc agatggagaa ggacattgcc atggagcaag agaggaatgc tcgctaccga 2100
acccccaaaa tcttggagcc cacagccttc caggaacccc cacccaagcc cagccgacct 2160
aagtacagac cccctccgca aaccaacctc ctggctccaa agctgcagtt ccaggttcct 2220
gagggtctgt gtgccagctc tcctacgctc accagcccta tggagtatcc atctcccgtt 2280
aactcactgc acaccccacc tctccaccgg cacaatgtct tcaaacgcca cagcatgcgg 2340
gaggaggact tcatccaacc cagcagccga gaagaggccc agcagctgtg ggaggctgaa 2400
aaggtcaaaa tgcggcaaat cctggacaaa cagcagaagc agatggtgga ggactaccag 2460
tggctcaggc aggaggagaa gtccctggac cccatggttt atatgaatga taagtcccca 2520
ttgacgccag agaaggaggt cggctacctg gagttcacag ggcccccaca gaagcccccg 2580
aggctgggcg cacagtccat ccagcccaca gctaacctgg accggactga tgacctggtg 2640
tacctcaatg tcatggagct ggtgcgggcc gtgctggagc tcaagaatga gctctgtcag 2700
ctgccccccg agggctacgt ggtggtggtg aagaatgtgg ggctgaccct gcggaagctc 2760
atcgggagcg tggatgatct cctgccttcc ttgccgtcat cttcacggac agagatcgag 2820
ggcacccaga aactgctcaa caaagacctg gcagagctca tcaacaagat gcggctggca 2880
cagcagaacg ccgtgacctc cctaagtgag gagtgcaaga ggcagatgct gacggcttca 2940
cacaccctgg ctgtggacgc caagaacctg ctcgacgctg tggaccaggc caaggttctg 3000
gccaatctgg cccacccacc tgcagagtga cggagggtgg gggccacctg cctgcgtctt 3060
ccgcccctgc ctgccatgta cctcccctgc cttgctgttg gtcatgtggg tcttccaggg 3120
ggaaggccaa ggggagtcac cttcccttgc cactttgcac gacgccctct ccccacccct 3180
acccctggct gtactgctca ggctgcagct ggacagaggg gactctgggc tatggacaca 3240
gggtgacggt gacaaagatg gctcagaggg ggactgctgc tgcctggcca ctgctcccta 3300
agccagcctg gtccatgcag ggggctcctg ggggtgggga ggtgtcacat ggtgccccta 3360
gctttatata tggacatggc aggccgattt gggaaccaag ctattccttt cccttcctct 3420
tcggccctca gatgtccctt gatgcacaga gaagctgggg aggagctttg ttttgggggt 3480
caggcagcca gtgagatgag ggatgggcct ggcattcttg tacagtgtat attgaaattt 3540
atttaatgtg agtttggtct ggactgacag catgtgccct cctgagggag gacctggggc 3600
acagtccagg aacaagctaa ttgggagtcc aggcacagga tgctgtgttg tcaacaaacc 3660
aagcatcagg gggaagaagc agagagatgc ggccaagata ggaccttggg ccaaatccgc 3720
tctcttcctg cccctctttc tctttcttcc tttactttcc cttgcttttc cctcttttct 3780
tactcctcct ctttctctcc ccaaccccca ttctcatctg cacccttctt ttctcatgtg 3840
tttgcataaa cattctttta acttctttct atttgacttg tggttgaatt aaaattgtcc 3900
catttgcttt gc 3912
<210> 2
<211> 24
<212> DNA
<213> Homo sapiens
<400> 2
acagtgtagg caacagcaca gaag 24
<210> 3
<211> 24
<212> DNA
<213> Homo sapiens
<400> 3
cgtgaagcgt cggaagttaa tgga 24
<210> 4
<211> 20
<212> DNA
<213> Mus musculus
<400> 4
ctcggggctc atgagctcaa 20
<210> 5
<211> 20
<212> DNA
<213> Mus musculus
<400> 5
tgctgtccgg aacatcctgg 20
<210> 6
<211> 3700
<212> DNA
<213> Mus musculus
<400> 6
cagtcaggag ccacattgtt tgcctaggct ggtaatcaag tctgagatgg ttgtgctgag 60
actgaaggga agacaccttc ttctgaaaag cattgtggtc cctgaacaca cacttctatt 120
gcagaaaggg gaaaaaatta atgtggccgt caagacctgt aagaaagact gtacccagga 180
caacaaggag aagttcatga gtgaggcagg taggcatccc ctggggagag gcccctgaga 240
tgctccagcc cccggaatgg gagctgggag gctgggaagg aagtccgagc agcaacggga 300
ccccaccccc cactcttgat tgcaaaaggt ctcttgtccc atgttccaca gctggggcag 360
gaaactgtgt ttctctttat gtgtctagga ggggaggctg tgttggtggt ggaacacttg 420
cctggcatgc ataaggtcct gtttgctcct cagcactggg gagagaaaag ccaagtgttt 480
tctgtatgac ctttaacctg cttcatctcc ctcgccctcc ttcagtgatc atgaagaatc 540
ttgaccaccc tcacatcgtg aagctgattg gcatcattga agaggaaccc acctggatta 600
tcatggaact gtatccttat ggggaggtaa gctcaggggc cttgataagg gacttcatgt 660
gacaaccggg ctgcggtgga gcaaggcatg ggaggcattt acagcaaagg gtttttccta 720
ttaagtgtct ttcctttaac tattaggtgg aggaattgcc gaaacatatt accatctttg 780
aatttgcccc tcaagggaat tgaagggtca gtggctatca gtagcctggt gccttggtgc 840
catgctggga cagaaagtca ttctggtgtc ctatctcttc ctgcagctgg gacactacct 900
ggaacgaaat aaaaactccc tgaaggtacc cactctggtc ctgtacaccc tacagatatg 960
caaagccatg gcctatctgg agagcatcaa ctgtgtgcac aggtaggagg ggtgtgagac 1020
tttaacctgg ttcccgagag ggagacagtg catagaattt aagagtgata tggtcctaag 1080
ccaggccccc agcccggtct gtgcctggca actgagagtg ctgggtactc cagactcaga 1140
tagtctccat gaaacatttc ctcaccggga aacttgaaag gtccaaggat actgagagga 1200
agtccaaagt ctgaggtctc actgtagctc tgtgccttct gctgcatcct ggcaagtgct 1260
gggggcgggg gctgccctct ggctcagagc tccagctcct ccactttgct ctctgcatga 1320
cacacagtct aggaagaagg aagtgggcta gagaagtgtc tggagcttcc tagggtcttt 1380
tggtctccac cctagttctc tggtgttcct gaaccacctt ctactttaac tatagctaca 1440
cgggtggctc ttccctgctc tccactggct ttgttctcat tcccgcatcg cattgtctga 1500
gtaggtgttg agctcatgag ccccgaggag actacactgg gatgctcacc ccaccacaga 1560
catggcctgt gtggctttga gcctgattct gtgcagagcc atggaggaat tacaaacaac 1620
tactccccgc atcccccact tcatccatca acttccttgg aaataccttc tcaatccagc 1680
ctgtgtccta gaacaaagtc acgaccaaca gaaacaagaa tcctttccct ttggaatcac 1740
attctagtgt gagggtcaaa tgggggtttc acacggcctg gggctaggga gatgtgctca 1800
caggtttcct ttatgtatct gaagctgtgt gttttaagaa tcatcctttt agtagcagaa 1860
actgcaggaa gtggcatggc ctgtgagaag caggagggcc ctttggggtt ggtgtgccac 1920
tatgaatgca gccggtccag gtcagcagat actcctcaag gagagctaga tacctatgtg 1980
ttttagaact tatactaccc tggtggcact ctcttcctgt cagaaacctc ttggtatgtt 2040
ccttcaggcc gatgcctggc tctctcagcg tgtaccaggg tcctgaggca gtgcctcata 2100
ttagccctcg attcctcttt cagggatatt gctgtccgga acatcctcgt cgcctctcgt 2160
gagtgtgtga agctggggga ctttgggctc tcccggtaca ttgaggacga agactattac 2220
aaaggtgagg gcttcccagt ggccagtatg gtttatagaa ctcaaaattc cagagcaagg 2280
aagttgggtt caggcagcct cggccagtgg tctctgaaga gtaggaatac aattctagat 2340
cttacatccc taaataatcc cttaaacctc tttgtcaaag cctctgtgac ccgtctaccc 2400
atcaaatgga tgtcccccga gtccatcaac ttccgccgct tcacaaccgc cagtgatgtc 2460
tggatgtttg gtgagtggcg attagaatcg ggtgggcctg ggaaagtgct gattctcatg 2520
tctgtggcct gagcaggagg gcagaagaga gaacaagggt cttacgtgtg cccctcaggt 2580
cccagtgtca ctgagaagtc ccaaaggttc agagagtcag agcagccatg acatgggcaa 2640
gggcccggtt gggataaact gggctcactc cctcgtaaag actcttgtca aaagcttgga 2700
ctacatcagc catccccagg gactgtgtgg gaaaggcttc atggagacct taaagaaagc 2760
tggccagctt cttttgatga ggaggctcaa gaagagagag aagccagctg atgtgatgga 2820
catttccgcc tgcagctatg tctgggaaac aacatcactg ctccccctga gcattctcca 2880
cttgaggaaa tgaaggctca gaggagccag gaccttggcc aaggacacag gcaacaagtg 2940
tcagtactag tattagaact ccttaaccct tgaagaagag gttttgtgtc ctacctgccc 3000
atgaggaagt tctgatcata cagataatag cctcctctgg atccccagca tcttagtagc 3060
tctacaggga gggttctaag gagcagattg tagttcatgt actataggta taggctgtag 3120
ttgtcttggc ctggaccatt gtgatgggca ggacctcaag ggagattgtg cctcctccag 3180
ttgggaccca ccagtggcca cagttatcgc tagggagagc aagctggtcc ctcccctgag 3240
tcttccctct ctgctctcag ctgtatgcat gtgggagatc ctcagctttg ggaagcagcc 3300
tttcttctgg ctcgaaaata aggatgtcat cggagtgctg gagaaagggg acaggctgcc 3360
caagcccgaa ctctgtccgc ctgtccttta cacactcatg actcgctgct gggactacga 3420
ccccagtgac cggccccgct tcacggagct tgtgtgcagc ctcaggtgag cataaggtgg 3480
ggaactctaa gtagaggatg acgctgcaga tgctgtgggg gtctccagcc ctgtgcagtg 3540
ggtatctttg ttcccctttg tcaggagagt tcgggttaaa gttggtgact tgatgcctaa 3600
tagtgacagg taaaatggca cttataccag aacttccttc tgatcagcaa gaggaaggtg 3660
ccctctcttc cagtagaagc acactgggtg gcagtgttgg 3700
<210> 7
<211> 23
<212> DNA
<213> Mus musculus
<400> 7
gcatcgcatt gtctgagtag gtg 23
<210> 8
<211> 20
<212> DNA
<213> Mus musculus
<400> 8
tgggaagccc tcacctttgt 20
<210> 9
<211> 20
<212> DNA
<213> Mus musculus
<400> 9
ggtggtggaa cacttgcctg 20
<210> 10
<211> 21
<212> DNA
<213> Mus musculus
<400> 10
ctcagacaat gcgatgcggg a 21
<210> 11
<211> 23
<212> DNA
<213> Mus musculus
<400> 11
gcatcgcatt gtctgagtag gtg 23
<210> 12
<211> 20
<212> DNA
<213> Mus musculus
<400> 12
gaaaggctgc ttcccaaagc 20
<210> 13
<211> 21
<212> DNA
<213> Mus musculus
<400> 13
ctccacccta gttctctggt g 21
<210> 14
<211> 20
<212> DNA
<213> Mus musculus
<400> 14
ccatggctct gcacagaatc 20
<210> 15
<211> 20
<212> DNA
<213> Mus musculus
<400> 15
ggtggtggaa cacttgcctg 20
<210> 16
<211> 21
<212> DNA
<213> Mus musculus
<400> 16
ctcagacaat gcgatgcggg a 21
<210> 17
<211> 21
<212> DNA
<213> Mus musculus
<400> 17
ggtccaggtc agcagatact c 21
<210> 18
<211> 20
<212> DNA
<213> Mus musculus
<400> 18
ggagggacca gcttgctctc 20
Claims (9)
1.一种原发性胆汁性胆管炎的新突变SNP位点,其特征在于,所述新突变SNP位点为人PTK2B基因外显子24,NM_173174.2,c.1679C→G,p.560,Pro(P)→Arg(R)。
2.一种用于鉴定权利要求1所述的原发性胆汁性胆管炎的新突变SNP位点的引物对,其特征在于,所述引物对的核苷酸序列如SEQ ID No.2-3所示。
3.一种用于鉴定权利要求1所述的原发性胆汁性胆管炎的新突变SNP位点的试剂盒,其特征在于,含有权利要求2所述的引物对。
4.如权利要求1所述的新突变SNP位点在制备原发性胆汁性胆管炎检测、诊断、治疗和/或预后产品中的应用。
5.如权利要求4所述的应用,其特征在于,所述产品为试剂盒,所述试剂盒包括用于扩增权利要求1所述的新突变SNP位点的引物对。
6.如权利要求5所述的应用,其特征在于,所述引物对的核苷酸序列如SEQ ID No.2-3所示。
7.如权利要求5或6所述的应用,其特征在于,所述试剂盒还包括dNTPs、Taq酶、Mg2+和/或PCR反应缓冲液。
8.一种原发性胆汁性胆管炎的动物模型的构建方法,其包括如下步骤:
1)设计PTK2b基因19号外显子靶向的sgRNA识别序列,构建sgRNA;
2)设计构建有PTK2B-P560R片段的供体质粒;
3)将Cas9 mRNA,sgRNA和供体共注射到小鼠受精卵,受精卵在交配后0.5天被移植到ICR雌性小鼠的输卵管中,移植后的19-21天F0代小鼠出生;
4)将携带有PTK2B基因点突变的F0代小鼠和C57BL/6J回交以得到杂合突变小鼠;
5)将携带有PTK2B基因点突变的F1代雌雄小鼠进行繁育,得到携带基因突变的后代小鼠。
9.如权利要求8所述的原发性胆汁性胆管炎的动物模型的构建方法,其特征在于,sgRNA的序列如SEQ ID No.4-5所示。
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WO2007095032A2 (en) * | 2006-02-09 | 2007-08-23 | Novartis Ag | Mutations and polymorphisms of ptk2b |
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