CN111100874A - 打靶载体及整合外源基因至小鼠dc-sign外显子7位点构建bac克隆的方法和应用 - Google Patents
打靶载体及整合外源基因至小鼠dc-sign外显子7位点构建bac克隆的方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,主要是一种打靶载体及整合外源基因至小鼠DC‑SIGN外显子7位点构建BAC克隆的方法和应用,一种靶向打靶载体序列如SEQ ID NO.14所示,命名为打靶载体pL451‑DCSIGN 5HA‑IRES‑DTR‑PEN‑DC SIGN 3HA。本发明提供了一个新的能实现靶向整合的位点序列,利用该位点可构建靶向插入外源基因的打靶载体,并验证了利用该打靶载体能高效整合外源基因至小鼠DC‑SIGN BAC克隆DC‑SIGN外显子7位点,后续得到的靶向插入外源基因的DC‑SIGN BAC,可以用于构建靶向插入外源基因的小鼠胚胎干细胞,从而为构建转基因细胞系以及小鼠建立了基础。
Description
技术领域
本发明属于生物技术领域,主要是一种打靶载体及整合外源基因至小鼠DC-SIGN外显子7位点构建BAC 克隆的方法和应用。
背景技术
通过同源重组将外源基因定点整合至靶细胞基因组上某一确定的位点,以达到定点修饰改造染色体上某一基因的目的,这种技术称为基因打靶技术。利用基因打靶技术将外源基因靶向整合至特定的染色体位点在基因疗法、细胞工程、遗传动物模型、基因功能研究及药物开发等中具有重要应用价值,而所有这些应用一方面依赖于转入基因功能的可靠性和可预测性,另一方面要求转入基因不影响内源基因和或其他调控因子的功能。树突状细胞特异性细胞间黏附分子-3结合非整合素因子(dendritic cell specificintercellular-adherison-molecular-3grabbing non-integr,DC-SIGN)是一种主要表达于树突状细胞(DC) 表面的模式识别受体,属于C型凝集素超分子家族。研究发现,当外周血中的单核细胞可以在GM-CSF 和IL-4的刺激下分化为DC,组织中处于稳态的DC能自我更新,而不需要单核细胞衍生DC的补充。有趣的是,单核细胞衍生DC表达DC-SIGN而组织中自我更新的DC不表达。所以DC-SIGN可以作为一个基因敲入的位点,使外源基因在单核细胞衍生DC中特异性表达,在其他细胞中不表达。而且我们把插入位点设置在DC-SIGN基因终止密码子之后,并利用IRES启动外源基因表达,最小化外源基因插入对细胞功能的影响。
细菌人工染色体(Bacterial artificial chromosome,BAC)是指一种以F质粒为基础建构而成的细菌染色体克隆载体。在基因打靶技术中,为了保证同源重组的效率需要长片段的同源臂,而直接克隆长片段的同源臂较为困难。作为一个基因保存的载体,BAC可以克隆约150kb的DNA且具有遗传背景简单的特点。我们可以将需要靶向插入的外源基因利用短同源臂先导入相应的BAC克隆,然后提取BAC进一步作为基因打靶的载体,以提高基因打靶的效率。
发明内容
本发明的目的在于克服现有技术存在的不足,而提供打靶载体及整合外源基因至DC-SIGN外显子7 位点构建靶向插入外源基因的小鼠DC-SIGN BAC克隆的方法和应用。
本发明的目的是通过如下技术方案来完成的。一种靶向打靶载体序列如SEQ IDNO.14所示,命名为打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DC SIGN 3HA;该载体能将外源基因IRES-DTR及抗性基因Neomycin靶向整合至DC-SIGN外显子7位点终止密码子之后。靶向整合后IRES-DTR由DC-SIGN 启动子调控被转录,外源基因DTR在IRES的调控下被翻译,且不影响DC-SIGN表达,抗性基因Neomycin 在PGK(哺乳动物)和EM7(大肠杆菌)的调控下被表达。如需要表达其它基因,只要将外源基因DTR替换成感兴趣的基因。
上述靶向打靶载体的制备方法包括如下步骤:
(1)以RP23-12K14 BAC DNA为模板,利用序列如SEQ ID NO.1所示的正向引物和序列如SEQ ID NO.2所示的反向引物进PCR,扩增出序列如SEQ ID NO.3所示的基因片段DCSIGN 5’侧同源臂 (DCSIGN5HA);
(2)将目的基因片段DCSIGN5HA利用酶切位点KpnI、SalI克隆至带阳性选择标记的载体 pL451-targeted-empty(counter-Selection BAC Modification Kit,Genebridges),得到中间载体 pL451-DCSIGN 5HA-PEN。
(3)以pLVX-EF1α-IRES-mCherry质粒(addgene)为模板,利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR,扩增出序列如SEQ IDNO.6所示的基因片段IRES;以pIRES-proHB EGF WT质粒(addgene)为模板,利用序列如SEQID NO.7所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行PCR,扩增出序列如SEQID NO.9所示的基因片段DTR;Overlap PCR 得到序列如SEQ ID NO.10所示的融合基因片段IRES-DTR;
(4)将目的基因片段IRES-DTR利用酶切位点SalI、EcoRI克隆至中间载体pL451-DCSIGN 5HA-PEN,得到中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN。
(5)以RP23-12K14BAC DNA为模板,利用序列如SEQ ID NO.11所示的正向引物和序列如SEQ ID NO.12所示的反向引物进行PCR,扩增出序列如SEQ ID NO.13所示的基因片段DC-SIGN3’侧同源臂 (DCSIGN3HA);
(6)将目的基因片段DCSIGN3HA利用酶切位点BamHI、NotI克隆至中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN,得到序列如SEQ ID NO.14所示的打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA。
上述靶向打靶载体可用于靶向整合外源基因至DC-SIGN外显子7位点构建小鼠DC-SIGN BAC克隆。
本发明还提供利用上述打靶载体,靶向整合外源基因至DC-SIGN外显子7位点,构建靶向插入外源基因的小鼠DC-SIGN-DTR BAC克隆的方法。具体步骤如下:
(1)电穿孔法将Red/ET表达质粒pRED/ET转化入DC-SIGN BAC克隆,在有氯霉素和四环素抗性的LB平板上筛选,挑取单菌落扩增,并用L-阿拉伯糖诱导Red/ET表达;
(2)打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA利用NotI酶切线性化,电穿孔法将线性化的载体转化入上述表达Red/ET的DC-SIGN BAC克隆,使IRES-DTR-PGK-EM7-Neo靶向插入 DC-SIGN BAC;
(3)成功插入IRES-DTR-PGK-EM7-Neo的DC-SIGN BAC克隆表达新霉素抗性,用有新霉素和氯霉素的LB平板筛选得BAC克隆DCSIGN-IRES-DTR-PEN;
(4)挑选单克隆,利用序列如SEQ ID NO.15所示的正向引物和序列如SEQ IDNO.16所示的反向引物作菌落PCR,成功插入IRES-DTR-PGK-EM7-Neo的DC-SIGN BAC克隆能够得到约1.4kb的条带。 SpeI酶切鉴定阳性BAC克隆可见4-6kb间多出2条带。
至此,本发明提供了靶向打靶载体及靶向整合外源基因至DC-SIGN外显子7位点,构建靶向插入外源基因的小鼠DC-SIGN BAC克隆的制备和检测方法。
本发明的有益效果为:
本发明提供了一种高效、安全靶向整合外源基因至小鼠DC-SIGN BAC克隆DC-SIGN外显子7位点的方法和应用实例。
本发明提供了靶向小鼠DC-SIGN外显子7位点的打靶载体左、右同源臂DNA序列信息及构建方法。
本发明提供了筛选与鉴定阳性克隆的方法和应用实例。
如背景技术所提到,DC-SIGN可以作为一个基因敲入的位点,使外源基因在单核细胞衍生DC中特异性表达并且不影响细胞功能,本发明提供了一个新的能实现靶向整合的位点序列,利用该位点可构建靶向插入外源基因的打靶载体,并验证了利用该打靶载体能高效整合外源基因至小鼠DC-SIGN BAC克隆 DC-SIGN外显子7位点,后续得到的靶向插入外源基因的DC-SIGN BAC,可以用于构建靶向插入外源基因的小鼠胚胎干细胞,从而为构建转基因细胞系以及小鼠建立了基础。
附图说明
图1:构建靶向小鼠DC-SIGN外显子7位点的打靶质粒流程示意图;
图2:携带抗性基因Neomycin的空载体pL451-targeted-empty的质粒结构示意图;
图3:携带外源基因IRES-DTR、抗性基因Neomycin和DC-SIGN两侧同源臂的打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DC SIGN 3HA质粒结构示意图;
图4:打靶载体打靶原理及同源重组插入DC-SIGN BAC后进行PCR筛选与鉴定的引物(黑色箭头)示意图;
图5:PCR筛选与鉴定靶向DC-SIGN外显子7位点的DC-SIGN BAC单克隆,正确靶向的单克隆出现约 1.4kb条带;
图6:酶切鉴定成功靶向DC-SIGN外显子7位点的DC-SIGN BAC单克隆,阳性克隆可见4-6kb间多出 2条带(黑色箭头)。
具体实施方式
下面将结合附图对本发明做详细的介绍:
实施例1:构建靶向整合外源基因至小鼠DC-SIGN外显子7位点的打靶载体
从genome.ucsc.edu网站检索并下载获得小鼠DC-SIGN基因组序列,结合小鼠DC-SIGN(Genbank No:NM_133238.5)序列,确定各外显子和内含子位置及序列。包含整个DC-SIGN基因的BAC Clone: RP23-12K14从Chieldren’hospotal Oakland ResearchInstitute购买获得,BAC DNA利用 HiPure Plasmid Filter Maxiprep Kit(Invitrogen)制备备用。
PCR扩增得目的基因片段DC-SIGN5’侧同源臂(DCSIGN5HA):以RP23-12K14 BACDNA为模板,利用正向引物DCSIGN-5HA-F(KpnI)-ACCACCGGTACCCTCCACATGCATATGTACACACAC(SEQ ID NO.1),反向引物DCSIGN-5HA-R(SalI)-AACAACGTCGACGACTGTGGAGATGGTGGAGG(SEQID NO.2)作PCR扩增5’侧同源臂DCSIGN5HA(SEQ ID NO.3)。下划线序列为酶切位点。
表1:PCR反应体系如下:
表2:PCR扩增条件如下:
将目的基因片段DCSIGN 5HA克隆至带阳性选择标记基因的载体pL451-targeted-empty:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA GelExtraction Kit(Axygen)回收。带 Neomycin阳性选择标记的载体pL451-targeted-empty(counter-Selection BAC Modification Kit, Genebridges)和回收后的DCSIGN 5HA经KpnI、SalI(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel ExtractionKit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化至大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示DCSIGN 5HA插入成功并命名为中间载体pL451-DCSIGN 5HA-PEN。
PCR扩增得目的基因片段IRES和DTR:以pLVX-EF1α-IRES-mCherry质粒(addgene)为模板,利用正向引物IRES-F(SalI)-ACCACCGTCGACGCCCCTCTCCCTCCCCCC(SEQ ID NO.4),反向引物 IRES-R-CATGGTTGTGGCAAGCTTATCATCGTG(SEQ ID NO.5)作PCR扩增基因片段IRES(SEQ ID NO.6);以pIRES-proHB EGF WT质粒(addgene)为模板,利用正向引物DTR-F-CACGATGATAAGCTT GCCACAACCATGAAGCTGCTGCCGTCG(SEQ ID NO.7),反向引物 DTR-R(EcoRI)-ACCACCGAATTCTTAGTGGGAATTAGTCATGCCC(SEQ ID NO.8)作PCR扩增 DTR(SEQ IDNO.9)。下划线序列为酶切位点。PCR反应体系同表1,PCR扩增条件同表2。
PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA GelExtraction Kit (Axygen)回收。IRES和DTR进行Overlap PCR,合成融合基因片段IRES-DTR(SEQ ID NO.10),具体步骤如下:
Overlap PCR反应体系如下:
Overlap PCR扩增条件为:
将目的基因片段IRES-DTR克隆至中间载体pL451-DCSIGN 5HA-PEN:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的IRES-DTR 和中间载体pL451-DCSIGN 5HA-PEN分别经SalI、EcoRI(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示IRES-DTR插入成功并命名为中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN。
PCR扩增目的基因片段DC-SIGN 3’侧同源臂(DCSIGN3HA):以RP23-12K14 BAC DNA为模板,利用正向引物DC-SIGN-3HA-F(BamHI)-ACCACCGGATCCCCAAAACCCTGCCAAATG(SEQ IDNO.11),反向引物DC-SIGN-3HA-R(NotI)-ACCACCGCGGCCGCTCTTGTCAAGGTTATCAATGGTCAC(SEQ ID NO.12)作PCR扩增3’侧同源臂DCSIGN 3HA(SEQ ID NO.13)。下划线序列为酶切位点。PCR反应体系同表1,PCR扩增条件同表2。
将目的基因片段DCSIGN 3HA克隆至中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA GelExtraction Kit(Axygen)回收。纯化后的DCSIGN 3HA和中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN分别经BamHI、(NEB)双酶切。酶切的 PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示DCSIGN 3HA插入成功并命名为打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA,序列如SEQ ID NO.14所示。至此靶向整合外源基因至小鼠DC-SIGN 外显子7位点的打靶质粒构建完成。
实施例2:利用打靶载体靶向整合外源基因至小鼠DC-SIGN BAC
电穿孔法将pRed/ET转化入包含整个DC-SIGN基因的大肠杆菌BAC Clone RP23-12K14:将包含整个DC-SIGN基因的大肠杆菌BAC CloneRP23-12K14接种于含氯霉素(15μg/mL)的LB平板,37℃培养 16h。挑选1个克隆于有1mL LB培养基(含15μg/mL氯霉素)的2mL离心管(盖子上戳一个洞使能够通气), 37℃200rpm培养过夜。取30μL培养过夜的大肠杆菌克隆于有1.4mL LB培养基(含15μg/mL氯霉素) 的2mL离心管,37℃1000rpm,培养2-3h扩增得对数期的大肠杆菌。扩增的大肠杆菌2℃11000rpm 离心30s,吸弃上清。大肠杆菌沉淀用1mL冰上预冷的ddH20清洗3遍,最后留下约30μL ddH2O重悬大肠杆菌,加入1μL pRed/ET(20ng/μL,counter-Selection BAC Modification Kit(Genebridges,USA)),轻弹混匀,冰上静置。转移大肠杆菌悬液至冰上预冷的1mm电穿孔管,利用Electroporator 2510进行电穿孔,1350V 10μF 600Ohms,将pRed/ET转化入BAC Clone RP23-212F14。用1mL无抗生素LB培养基重悬大肠杆菌并转移至2mL离心管,30℃1000rpm培养70min使抗性基因表达。
L-阿拉伯糖诱导Red/ET表达:取100μL转入pRed/ET并诱导抗性基因表达的大肠杆菌于含四环素 (3μg/mL)、氯霉素(15μg/mL)的LB平板,涂布均匀,30℃避光培养16h。挑取1个单克隆于有1mL LB培养基(含3μg/mL四环素,15μg/mL氯霉素)的2mL盖子戳洞的离心管,30℃200rpm培养过夜。取30μL大肠杆菌于1个有1mL LB培养基(含3μg/mL四环素,15μg/mL氯霉素)的2mL盖子戳洞的离心管,30℃1100rpm 培养2h,直到OD600大约0.3。加入50μL L-阿拉伯糖至终浓度为0.3-0.4%,37℃200rpm培养45-60min,诱导Red/ET表达。
电穿孔法将线性化的打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA转化入表达Red/ET的BAC Clone RP23-12K14:表达Red/ET的大肠杆菌进行离心,2℃11000rpm30s,吸弃上清。大肠杆菌沉淀用1mL冰上预冷的ddH20清洗3遍,最后留下约30μL ddH2O重悬大肠杆菌,加入1-2μl (100-200ng)用NotI(NEB)酶切线性化的打靶载体pL451-DCSIGN5HA-IRES-DTR-PEN-DCSIGN 3HA,轻弹混匀,冰上静置。转移大肠杆菌悬液至冰上预冷的1mm电穿孔管,利用Electroporator 2510进行电穿孔,1350V 10μF 600Ohms,将线性化的打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA转化入BAC CloneRP23-12K14。用1mL无抗生素LB培养基重悬大肠杆菌并转移至2mL离心管,30℃1000rpm培养70min使IRES-DTR-PGK-EM7-Neo同源重组插入 DC-SIGN BAC,并表达新霉素抗性基因。取100μL大肠杆菌于含四环素(3μg/mL)、氯霉素(15μg/mL)、新霉素(15μg/mL)的LB平板,涂布均匀,30℃培养大于20h,得到插入IRES-DTR-PGK-EM7-Neo的大肠杆菌克隆,命名为BAC CloneDCSIGN-IRES-DTR-PEN。
实施例3:外源基因靶向整合至小鼠DC-SIGN外显子7位点的BAC克隆的筛选与鉴定
菌落PCR筛选靶向插入IRES-DTR-PGK-EM7-Neo的大肠杆菌BAC Clone:挑选19个单克隆分别于有 10μl ddH2O的1.5mL离心管,吹打混匀。取1μl菌液作为模板,利用正向引物GHpA-F-TTCTGAGGCGGAAAGAACC(SEQ ID NO.15)和反向引物 DCSIGN-R-CAAAAGGACAGGCCCTAGAA(SEQ ID NO.16)作菌落PCR。剩余菌液加入含5mL LB培养基(含15μg/mL氯霉素,15μg/mL新霉素)的15mL离心管,37℃220rpm培养16h,pRed/ET在37℃时会丢失。
菌落PCR反应体系如下:
菌落PCR反应条件如下:
PCR产物经1%琼脂糖凝胶电泳鉴定,靶向插入IRES-DTR-PGK-EM7-Neo至DC-SIGN外显子7位点的大肠杆菌BAC阳性克隆可见约1.4kb条带,如附图5所示。
酶切鉴定阳性BAC克隆:过夜扩增的阳性BAC克隆菌液取4mL,用Counter-Selection(Advanced)BAC Modification Kit protocol version 3.0(Gene Bridges,USA)提取BAC。阳性克隆BAC经SpeI(NEB)酶切后, 1%琼脂糖凝胶电泳鉴定,阳性克隆在4-6kb会多出2条条带,如附图6所示。PCR和酶切鉴定均正确的靶向插入外源基因的阳性BAC克隆剩余菌液离心后去上清,用20%甘油的LB培养基重悬,-80℃保存。
可以理解的是,对本领域技术人员来说,对本发明的技术方案及发明构思加以等同替换或改变都应属于本发明所附的权利要求的保护范围。
序列表
<110>浙江大学医学院附属第一医院
<120>打靶载体及整合外源基因至小鼠DC-SIGN外显子7位点构建BAC克隆的方法和应用
<160>
<170>PatentIn version 3.3
<210> 1
<211> 36
<212> DNA
<213>引物DCSIGN-5HA-F(KpnI)
<400> 1
ACCACCGGTA CCCTCCACAT GCATATGTAC ACACAC
<210> 2
<211> 32
<212> DNA
<213>引物DCSIGN -5HA-R(SalI)
<400> 2
AACAACGTCG ACGACTGTGG AGATGGTGGA GG
<210> 3
<211>1025
<212> DNA
<213>基因片段DCSIGN5HA
<400> 3
ACCACCGGTA CCCTCCACAT GCATATGTAC ACACACACAC ACACACACAC ACACACGAAC 60
ACAATCACAC AGAGATCAAA AATTACTGTT GCCATTAACA ATATAACTTG TAGGTGGAAT 120
TGGAGAGGTT GCTCAGTGGT TAAGAACCTA GGAGTCCTTA ATTCCAGATC TGCAAAAAAA 180
CTGAAACTTC CAGATGACAC AAGTTTGGTT CTCAGTTCTC ACATCAATTA GCTCACAACC 240
ACCTGTAATT CCAGCTCTGC ATGCACCTAT ACTCACATAT GCATACATGC ACCTGCACAT 300
TCAAGGACAA TAAACCCACA CGTGTGTATG TACACACACA CACACACACA CACACACACA 360
CACTCCACAA AAAGAAAAGA ATATGCCCAG AGCTTGTTGT ACTCAAAGGT CCAAAAAGAA 420
GGAATTTATA TCACCACTTG AGGCAGGAGA CCAAGGCCCT GACCTCTAGA TGTGAAGGGA 480
TCAGGAGATT TGCACAAAGG CCTTGGTTGA CATGTCAAGG TAAGAAATAG ATGATGTGGG 540
TCAAGGAGGC TTAGATGGGA TCTTTTTAAA GTGTGTTAGT GTGTTCTAGG TGCTGTCTTT 600
CCTGGCTTGC CTTGTATTGA TGTAGTAAAC ACCATGACTA AAAGCAGTGT GGGGAGGAAT 660
GGGTTTATTT CCTCTTACAA CTCCCAGGTC ACACTCCATC ACTGAGGGAA GCCAGGGCAG 720
GGGCTTAAGA CAGGAACTAC AGCAGAAGCC ACAGAGGAAC ACAGCTCACT GTGCTTTCCA 780
TGGCTTAAAG ACTCTGTCTG TGACAGAATC TCGTGTCTCC TTGTCTTTTC AGTTTCATGA 840
AGTATTGGAG TAAAGGAGAA CCTAACAACC TGGGAGAGGA AGACTGTGCA GAGTTCAGAG 900
ATGACGGCTG GAATGACACC AAATGTACTA ACAAGAAATT CTGGATCTGC AAAAAGCTTT 960
CAACTTCCTG CCCTAGCAAG TGATGGCCAA CTCCCTCCAC CATCTCCACA GTCGTCGACG 1020
TTGTT
<210> 4
<211> 30
<212> DNA
<213>引物IRES-F(SalI)
<400> 4
ACCACCGTCG ACGCCCCTCT CCCTCCCCCC
<210> 5
<211> 27
<212> DNA
<213>引物IRES-R
<400> 5
CATGGTTGTG GCAAGCTTAT CATCGTG
<210> 6
<211> 602
<212> DNA
<213>基因片段IRES
<400> 6
ACCACCGTCG AGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA 60
ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 120
TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 180
TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 240
TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG 300
CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 360
ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCTCCTCAAG 420
CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT 480
GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 540
CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAAGCTTG CCACAACCAT 600
GA
<210> 7
<211> 42
<212> DNA
<213>引物DTR-F
<400> 7
CACGATGATA AGCTTGCCAC AACCATGAAG CTGCTGCCGT CG
<210> 8
<211> 34
<212> DNA
<213>引物DTR-R(EcoRI)
<400> 8
ACCACCGAATTCTTAGTGGG AATTAGTCAT GCCC
<210> 9
<211> 663
<212> DNA
<213>基因片段DTR
<400> 9
CACGATGATA AGCTTGCCAC AACCATGAAG CTGCTGCCGT CGGTGGTGCT GAAGCTCTTT 60
CTGGCTGCAG TTCTCTCGGC ACTGGTGACT GGCGAGAGCC TGGAGCGGCT TCGGAGAGGG 120
CTAGCTGCTG GAACCAGCAA CCCGGACCCT CCCACTGTAT CCACGGACCA GCTGCTACCC 180
CTAGGAGGCG GCCGGGACCG GAAAGTCCGT GACTTGCAAG AGGCAGATCT GGACCTTTTG 240
AGAGTCACTT TATCCTCCAA GCCACAAGCA CTGGCCACAC CAAACAAGGA GGAGCACGGG 300
AAAAGAAAGA AGAAAGGCAA GGGGCTAGGG AAGAAGAGGG ACCCATGTCT TCGGAAATAC 360
AAGGACTTCT GCATCCATGG AGAATGCAAA TATGTGAAGG AGCTCCGGGC TCCCTCCTGC 420
ATCTGCCACC CGGGTTACCA TGGAGAGAGG TGTCATGGGC TGAGCCTCCC AGTGGAAAAT 480
CGCTTATATA CCTATGACCA CACAACCATC CTGGCCGTGG TGGCTGTGGT GCTGTCATCT 540
GTCTGTCTGC TGGTCATCGT GGGGCTTCTC ATGTTTAGGT ACCATAGGAG AGGAGGTTAT 600
GATGTGGAAA ATGAAGAGAA AGTGAAGTTG GGCATGACTA ATTCCCACTA AGAATTCGGT 660
GGT
<210> 10
<211> 1238
<212> DNA
<213>基因片段IRES-DTR
<400> 10
ACCACCGTCG ACGCCCCTCT CCCTCCCCCC CCCCTAACGT TACTGGCCGA AGCCGCTTGG 60
AATAAGGCCG GTGTGCGTTT GTCTATATGT TATTTTCCAC CATATTGCCG TCTTTTGGCA 120
ATGTGAGGGC CCGGAAACCT GGCCCTGTCT TCTTGACGAG CATTCCTAGG GGTCTTTCCC 180
CTCTCGCCAA AGGAATGCAA GGTCTGTTGA ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG 240
CTTCTTGAAG ACAAACAACG TCTGTAGCGA CCCTTTGCAG GCAGCGGAAC CCCCCACCTG 300
GCGACAGGTG CCTCTGCGGC CAAAAGCCAC GTGTATAAGA TACACCTGCA AAGGCGGCAC 360
AACCCCAGTG CCACGTTGTG AGTTGGATAG TTGTGGAAAG AGTCAAATGG CTCTCCTCAA 420
GCGTATTCAA CAAGGGGCTG AAGGATGCCC AGAAGGTACC CCATTGTATG GGATCTGATC 480
TGGGGCCTCG GTGCACATGC TTTACATGTG TTTAGTCGAG GTTAAAAAAC GTCTAGGCCC 540
CCCGAACCAC GGGGACGTGG TTTTCCTTTG AAAAACACGA TGATAAGCTT GCCACAACCA 600
TGAAGCTGCT GCCGTCGGTG GTGCTGAAGC TCTTTCTGGC TGCAGTTCTC TCGGCACTGG 660
TGACTGGCGA GAGCCTGGAG CGGCTTCGGA GAGGGCTAGC TGCTGGAACC AGCAACCCGG 720
ACCCTCCCAC TGTATCCACG GACCAGCTGC TACCCCTAGG AGGCGGCCGG GACCGGAAAG 780
TCCGTGACTT GCAAGAGGCA GATCTGGACC TTTTGAGAGT CACTTTATCC TCCAAGCCAC 840
AAGCACTGGC CACACCAAAC AAGGAGGAGC ACGGGAAAAG AAAGAAGAAA GGCAAGGGGC 900
TAGGGAAGAA GAGGGACCCA TGTCTTCGGA AATACAAGGA CTTCTGCATC CATGGAGAAT 960
GCAAATATGT GAAGGAGCTC CGGGCTCCCT CCTGCATCTG CCACCCGGGT TACCATGGAG 1020
AGAGGTGTCA TGGGCTGAGC CTCCCAGTGG AAAATCGCTT ATATACCTAT GACCACACAA 1080
CCATCCTGGC CGTGGTGGCT GTGGTGCTGT CATCTGTCTG TCTGCTGGTC ATCGTGGGGC 1140
TTCTCATGTT TAGGTACCAT AGGAGAGGAG GTTATGATGT GGAAAATGAA GAGAAAGTGA 1200
AGTTGGGCAT GACTAATTCC CACTAAGAATTCGGTGGT
<210> 11
<211>30
<212> DNA
<213>引物DCSIGN-3HA-F(BamHI)
<400> 11
ACCACCGGAT CCCCAAAACC CTGCCAAATG
<210> 12
<211>39
<212> DNA
<213>引物DCSIGN-3HA-R(NotI)
<400> 12
ACCACCGCGG CCGCTCTTGT CAAGGTTATC AATGGTCAC
<210> 13
<211>112
<212> DNA
<213>基因片段DCSIGN 3HA
<400> 13
ACCACCGGAT CCCCAAAACC CTGCCAAATG GCAGAACTTT ACCCATAGCT ATGCCAGTTT 60
ATTCTACTTG TCTGTGACCA TTGATAACCT TGACAAGAGC GGCCGCGTTG TT
<210> 14
<211> 7085
<212> DNA
<213>打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN- DCSIGN 3HA
<400> 14
CTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 60
ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 120
GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 180
CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC 240
CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG 300
CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA 360
AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC 420
CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCC CATTCGCCAT TCAGGCTGCG 480
CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TGGCGAAAGG 540
GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CACGACGTTG 600
TAAAACGACG GCCAGTGAAT TGTAATACGA CTCACTATAG GGCGAATTGG GTACCCTCCA 660
CATGCATATG TACACACACA CACACACACA CACACACACG AACACAATCA CACAGAGATC 720
AAAAATTACT GTTGCCATTA ACAATATAAC TTGTAGGTGG AATTGGAGAG GTTGCTCAGT 780
GGTTAAGAAC CTAGGAGTCC TTAATTCCAG ATCTGCAAAA AAACTGAAAC TTCCAGATGA 840
CACAAGTTTG GTTCTCAGTT CTCACATCAA TTAGCTCACA ACCACCTGTA ATTCCAGCTC 900
TGCATGCACC TATACTCACA TATGCATACA TGCACCTGCA CATTCAAGGA CAATAAACCC 960
ACACGTGTGT ATGTACACAC ACACACACAC ACACACACAC ACACACTCCA CAAAAAGAAA 1020
AGAATATGCC CAGAGCTTGT TGTACTCAAA GGTCCAAAAA GAAGGAATTT ATATCACCAC 1080
TTGAGGCAGG AGACCAAGGC CCTGACCTCT AGATGTGAAG GGATCAGGAG ATTTGCACAA 1140
AGGCCTTGGT TGACATGTCA AGGTAAGAAA TAGATGATGT GGGTCAAGGA GGCTTAGATG 1200
GGATCTTTTT AAAGTGTGTT AGTGTGTTCT AGGTGCTGTC TTTCCTGGCT TGCCTTGTAT 1260
TGATGTAGTA AACACCATGA CTAAAAGCAG TGTGGGGAGG AATGGGTTTA TTTCCTCTTA 1320
CAACTCCCAG GTCACACTCC ATCACTGAGG GAAGCCAGGG CAGGGGCTTA AGACAGGAAC 1380
TACAGCAGAA GCCACAGAGG AACACAGCTC ACTGTGCTTT CCATGGCTTA AAGACTCTGT 1440
CTGTGACAGA ATCTCGTGTC TCCTTGTCTT TTCAGTTTCA TGAAGTATTG GAGTAAAGGA 1500
GAACCTAACA ACCTGGGAGA GGAAGACTGT GCAGAGTTCA GAGATGACGG CTGGAATGAC 1560
ACCAAATGTA CTAACAAGAA ATTCTGGATC TGCAAAAAGC TTTCAACTTC CTGCCCTAGC 1620
AAGTGATGGC CAACTCCCTC CACCATCTCC ACAGTCGTCG ACGCCCCTCT CCCTCCCCCC 1680
CCCCTAACGT TACTGGCCGA AGCCGCTTGG AATAAGGCCG GTGTGCGTTT GTCTATATGT 1740
TATTTTCCAC CATATTGCCG TCTTTTGGCA ATGTGAGGGC CCGGAAACCT GGCCCTGTCT 1800
TCTTGACGAG CATTCCTAGG GGTCTTTCCC CTCTCGCCAA AGGAATGCAA GGTCTGTTGA 1860
ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG CTTCTTGAAG ACAAACAACG TCTGTAGCGA 1920
CCCTTTGCAG GCAGCGGAAC CCCCCACCTG GCGACAGGTG CCTCTGCGGC CAAAAGCCAC 1980
GTGTATAAGA TACACCTGCA AAGGCGGCAC AACCCCAGTG CCACGTTGTG AGTTGGATAG 2040
TTGTGGAAAG AGTCAAATGG CTCTCCTCAA GCGTATTCAA CAAGGGGCTG AAGGATGCCC 2100
AGAAGGTACC CCATTGTATG GGATCTGATC TGGGGCCTCG GTGCACATGC TTTACATGTG 2160
TTTAGTCGAG GTTAAAAAAC GTCTAGGCCC CCCGAACCAC GGGGACGTGG TTTTCCTTTG 2220
AAAAACACGA TGATAATATG GCCACAACCA TGAAGCTGCT GCCGTCGGTG GTGCTGAAGC 2280
TCTTTCTGGC TGCAGTTCTC TCGGCACTGG TGACTGGCGA GAGCCTGGAG CGGCTTCGGA 2340
GAGGGCTAGC TGCTGGAACC AGCAACCCGG ACCCTCCCAC TGTATCCACG GACCAGCTGC 2400
TACCCCTAGG AGGCGGCCGG GACCGGAAAG TCCGTGACTT GCAAGAGGCA GATCTGGACC 2460
TTTTGAGAGT CACTTTATCC TCCAAGCCAC AAGCACTGGC CACACCAAAC AAGGAGGAGC 2520
ACGGGAAAAG AAAGAAGAAA GGCAAGGGGC TAGGGAAGAA GAGGGACCCA TGTCTTCGGA 2580
AATACAAGGA CTTCTGCATC CATGGAGAAT GCAAATATGT GAAGGAGCTC CGGGCTCCCT 2640
CCTGCATCTG CCACCCGGGT TACCATGGAG AGAGGTGTCA TGGGCTGAGC CTCCCAGTGG 2700
AAAATCGCTT ATATACCTAT GACCACACAA CCATCCTGGC CGTGGTGGCT GTGGTGCTGT 2760
CATCTGTCTG TCTGCTGGTC ATCGTGGGGC TTCTCATGTT TAGGTACCAT AGGAGAGGAG 2820
GTTATGATGT GGAAAATGAA GAGAAAGTGA AGTTGGGCAT GACTAATTCC CACTAAGAAT 2880
TCCGAAGTTC CTATTCTCTA GAAAGTATAG GAACTTCAGG TCTGAAGAGG AGTTTACGTC 2940
CAGCCAAGCT AGCTTGGCTG CAGGTCGTCG AAATTCTACC GGGTAGGGGA GGCGCTTTTC 3000
CCAAGGCAGT CTGGAGCATG CGCTTTAGCA GCCCCGCTGG GCACTTGGCG CTACACAAGT 3060
GGCCTCTGGC CTCGCACACA TTCCACATCC ACCGGTAGGC GCCAACCGGC TCCGTTCTTT 3120
GGTGGCCCCT TCGCGCCACC TTCTACTCCT CCCCTAGTCA GGAAGTTCCC CCCCGCCCCG 3180
CAGCTCGCGT CGTGCAGGAC GTGACAAATG GAAGTAGCAC GTCTCACTAG TCTCGTGCAG 3240
ATGGACAGCA CCGCTGAGCA ATGGAAGCGG GTAGGCCTTT GGGGCAGCGG CCAATAGCAG 3300
CTTTGCTCCT TCGCTTTCTG GGCTCAGAGG CTGGGAAGGG GTGGGTCCGG GGGCGGGCTC 3360
AGGGGCGGGC TCAGGGGCGG GGCGGGCGCC CGAAGGTCCT CCGGAGGCCC GGCATTCTGC 3420
ACGCTTCAAA AGCGCACGTC TGCCGCGCTG TTCTCCTCTT CCTCATCTCC GGGCCTTTCG 3480
ACCTGCAGCC TGTTGACAAT TAATCATCGG CATAGTATAT CGGCATAGTA TAATACGACA 3540
AGGTGAGGAA CTAAACCATG GGATCGGCCA TTGAACAAGA TGGATTGCAC GCAGGTTCTC 3600
CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT 3660
CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG 3720
ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA 3780
CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC 3840
TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA 3900
AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC 3960
CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC 4020
TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG 4080
CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT 4140
GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC 4200
TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC 4260
TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC 4320
AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGGGGATCAA TTCTCTAGAG 4380
CTCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC 4440
CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG 4500
AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATTCT GGGGGGTGGG GTGGGGCAGG 4560
ACAGCAAGGG GGAGGATTGG GAAGACAATA GCAGGCATGC TGGGGATGCG GTGGGCTCTA 4620
TGGCTTCTGA GGCGGAAAGA ACCAGCTGGG GCTCGACTAG AGCTTGCGGA ACCCTTCGAA 4680
GTTCCTATTC TCTAGAAAGT ATAGGAACTT CATCAGTCAG GTACATAATA TAACTTCGTA 4740
TAATGTATGC TATACGAAGT TATTAGGTGG ATCCCCAAAA CCCTGCCAAA TGGCAGAACT 4800
TTACCCATAG CTATGCCAGT TTATTCTACT TGTCTGTGAC CATTGATAAC CTTGACAAGA 4860
GCGGCCGCCA CCGCGGTGGA GCTCCAGCTT TTGTTCCCTT TAGTGAGGGT TAATTTCGAG 4920
CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC 4980
ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA 5040
ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA 5100
GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC 5160
CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC 5220
TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT 5280
GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT 5340
CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG 5400
AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC 5460
TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT 5520
GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA 5580
GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA 5640
TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA 5700
CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA 5760
CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT 5820
CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT 5880
TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT 5940
CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT 6000
GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC 6060
AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC 6120
ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA 6180
GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA 6240
CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG 6300
CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC 6360
TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT 6420
CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG 6480
GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT 6540
CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA 6600
TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA 6660
GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA 6720
TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG 6780
GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC 6840
ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG 6900
AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT 6960
CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT 7020
ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT 7080
GCCAC
<210> 15
<211>19
<212> DNA
<213>引物GHpA-F
<400> 15
TTCTGAGGCG GAAAGAACC
<210> 16
<211> 20
<212> DNA
<213>引物DCSIGN-R
<400> 16
CAAAAGGACA GGCCCTAGAA
Claims (4)
1.一种靶向打靶载体,其特征在于,所述靶向打靶载体序列如SEQ ID NO.14所示,命名为载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DC SIGN 3HA。
2.一种靶向打靶载体的制备方法,其特征在于:所述方法包括如下步骤:
(1)以RP23-12K14 BAC DNA为模板,利用序列如SEQ ID NO.1所示的正向引物和序列如SEQ ID NO.2所示的反向引物进PCR,扩增出序列如SEQ ID NO.3所示的基因片段DCSIGN 5’侧同源臂;
(2)将目的基因片段DCSIGN 5HA利用酶切位点KpnI、SalI克隆至带阳性选择标记的载体pL451-targeted-empty,得到中间载体pL451-DCSIGN 5HA-PEN;
(3)以pLVX-EF1α-IRES-mCherry质粒为模板,利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR,扩增出序列如SEQ ID NO.6所示的基因片段IRES;以pIRES-proHB EGF WT质粒为模板,利用序列如SEQ ID NO.7所示的正向引物和序列如SEQ ID NO.8所示的反向引物进行PCR,扩增出序列如SEQ ID NO.9所示的基因片段DTR;Overlap PCR得到序列如SEQ ID NO.10所示的融合基因片段IRES-DTR;
(4)将目的基因片段IRES-DTR利用酶切位点SalI、EcoRI克隆至中间载体pL451-DCSIGN5HA-PEN,得到中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN;
(5)以RP23-12K14 BAC DNA为模板,利用序列如SEQ ID NO.11所示的正向引物和序列如SEQ ID NO.12所示的反向引物进行PCR,扩增出序列如SEQ ID NO.13所示的基因片段DC-SIGN 3’侧同源臂;
(6)将目的基因片段DCSIGN 3HA利用酶切位点BamHI、NotI克隆至中间载体pL451-DCSIGN 5HA-IRES-DTR-PEN,得到序列如SEQ ID NO.14所示的打靶载体pL451-DCSIGN 5HA-IRES-DTR-PEN-DCSIGN 3HA。
3.一种靶向打靶载体在靶向整合外源基因至小鼠DC-SIGN外显子7位点构建BAC克隆的应用。
4.一种靶向打靶载体在靶向整合外源基因至小鼠DC-SIGN外显子7位点构建BAC克隆的方法,其特征在于,靶向整合外源基因的大肠杆菌BAC Clone构建方法包括以下步骤:
(1)电穿孔法将Red/ET表达质粒pRED/ET转化入DC-SIGN BAC克隆,并用L-阿拉伯糖诱导Red/ET表达;
(2)电穿孔法将酶切线性化的打靶载体转化入上述表达Red/ET的DC-SIGN BAC克隆,使IRES-DTR-PGK-EM7-Neo靶向插入DC-SIGN BAC;
(3)成功插入IRES-DTR-PGK-EM7-Neo的DC-SIGN BAC克隆表达新霉素抗性,用有新霉素和氯霉素的LB平板筛选得靶向整合外源基因至DC-SIGN外显子7位点的BAC克隆DCSIGN-IRES-DTR-PEN;
(4)挑选单克隆,利用序列如SEQ ID NO.15所示的正向引物和序列如SEQ ID NO.16所示的反向引物作菌落PCR,成功插入IRES-DTR-PGK-EM7-Neo的DC-SIGN BAC克隆能够得到1.4kb的条带。SpeI酶切鉴定阳性BAC克隆可见4-6kb间多出2条条带。
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