CN100510082C - 一种淋巴组织特异性基因表达调控序列 - Google Patents
一种淋巴组织特异性基因表达调控序列 Download PDFInfo
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Abstract
一种淋巴组织特异性基因表达调控序列及其构建方法，本发明涉及基因表达调控和基因工程领域，提供了用于淋巴细胞中特异性高表达的基因调控DNA序列。具有马立克氏病病毒编码的vIL8基因淋巴组织特异性基因表达正调控的启动子-470-+10的DNA序列。人们可用该基因序列构建相应的表达载体，驱动目的基因在靶器官组织中的特异性表达，用这些调控DNA序列在特定组织，调控一些抑制肿瘤的基因，包括有调理作用或诱导凋亡、抑制细胞生长信号传导，从而达到抑制、消灭肿瘤的目的，用这些调控DNA序列进行免疫增强和调节的药物研究与开发。
Description
技术领域
本发明涉及基因表达调控和基因工程领域，提供了用于淋巴细胞特异性高表达的基因调控的启动子DNA序列。更具体地，本发明提供了来源于马立克氏病病毒(Marek’s disease virus，MDV)的淋巴组织特异性启动子序列。
背景技术
近30年来，恶性淋巴瘤的发病率逐年上升。非霍奇金淋巴瘤(non-Hodgkin’slymphoma，NHL)在世界范围内的发病率几乎增长了一倍。人口老龄化，HIV感染和环境污染可能是其发病率增长的原因。尽管它对化疗非常敏感，新的化疗药物及化疗方案也不断涌现，但是其远期生存率仍徘徊在40％-50％之间。近20年来，随着生物学的发展，生物治疗在淋巴瘤治疗中的地位正日益引起关注，并成为其综合治疗的重要组成部分。而生物治疗中最引人注目的方面是淋巴瘤的靶向治疗。现在，生物治疗领域的靶向治疗中最主要的是单克隆抗体治疗。但是好的单克隆抗体要具有与靶抗原高亲和力等要求。而且单克隆抗体治疗有其不利因素：如靶抗原表达的改变，抗原抗体的可逆性结合，大肿块或肿瘤血供不良时，抗体难以到达肿瘤组织，循环池中大量游离靶抗原造成抗体被清除等等。
近年来，人们在寻找一些新方法进行肿瘤的生物靶向治疗。组织特异性启动子由于其在不同的细胞中的活性差异很大，因此可驱动目的基因在靶器官组织中的特异性表达。而且，启动子引起的组织特异性表达可降低免疫反应，并避免了对其他器官的副作用，可以很好的实现基因治疗的靶向性，从而成为生物治疗研究领域的新的热点。现已发现一系列组织特异性启动子，并开展了相关的研究，如：前列腺特异性启动子PSA启动子，卵巢特异性启动子OSP-1，肝脏特异性启动子小鼠白蛋白启动子，骨组织特异性启动子骨钙蛋白启动子，等等。人们可构建相应的表达载体，用这些启动子在特定组织调控一些抑制肿瘤的基因，包括有调理作用或诱导凋亡、抑制细胞生长信号传导达到抑制、消灭肿瘤的目的的一些基因，如：胞苷脱氨酶(CD)，多肿瘤抑制因子(MTS1)，一氧化氮合酶(NOS)，一些细胞因子类(TNF、IL-2、GM-CSF)，等等。这些启动子的发现和进一步构建转基因动物模型和建立基因治疗载体治疗癌症中具有很好的应用前景。如果能利用一些启动子有针对的在淋巴组织中表达一些抗肿瘤的基因，进而开发出针对淋巴瘤组织特异性的治疗药物，应该说这是一条研制抗淋巴瘤药物的新途径。
发明内容
本发明的目的是提供一种淋巴组织特异性基因表达调控序列(命名为pGL3-PvIL8)。它来源于鸡马立克氏病病毒编码的vIL8基因的一个淋巴细胞特异性正调控序列。
本发明之淋巴组织特异性基因表达调控序列(pGL3-PvIL8)具有马立克氏病病毒编码的vIL8基因淋巴组织特异性基因表达正调控的启动子-470-+10的DNA序列，其核苷酸DNA序列为：
gtgtgtagag ggcgcatgcg tactgccgta agcggtctgc gcatgcgttt tggactgcgc 60
atgcgttttg tgtgtagagg gcgcatgcgt actgcagtaa gtggtctgcg catgcgggtc 120
gtgtgtagag ggcgcatgcg tactgcaaaa tttattgtgg tcctggtttt ctgctatgca 180
ggggtcgtgg gaattttctc atatacattt aagggtcctc atagtcttgt gttccagtat 240
tatcaccttt tgatgcaaac tcctgttcat tttagtgttt cgatgctata gtttatgccc 300
catcgaatgg aaattttcaa taaacaaaat gcatctcacg gtcgtggaat tttaagttgg 360
gggtctccaa tatcacgtgt tggtggagac ccaataacag ggaaatcgcc cgaggcatta 420
cgggtacggt tccatggata tataatgcag ggggtgtggg tttgatgagc agttggggcg 480。
本发明为淋巴组织特异性正调控序列，由480个核苷酸组成的DNA片段。
序列1的5’端第10-21位、18-29位、87-98位、95-106位、258-269位核苷酸为CRE(cAMP反应元件)结合位点、自序列1的5’端第189-198位核苷酸为NF-κB结合位点、自序列1的5’端第185-197位核苷酸为Ikaros结合位点。
本发明的调控序列，在鸡胚法氏囊细胞(CEB，一种分化中的鸡B细胞)中具有组织特异性和高效转录活性。实验表明，本发明的启动子在CEB淋巴细胞中的转录活性高于其在CEF(鸡胚成纤维细胞)中的转录活性。可以预见，本发明的调控序列在构建转基因动物模型和建立基因治疗载体治疗淋巴瘤中具有很好的应用价值。
另，以下几种序列也具有正调控功能：
1、有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列片段；
2、遗传变体或缺失型有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列的片段；
3、序列的遗传变体或缺失型有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列；
4、序列至少60％与所述启动子-470-+10的DNA序列同源性；
5、序列至少有150个碱基对与所述启动子-470-+10的DNA序列同源性。
附图说明
图1为淋巴组织特异性基因表达调控序列的构建示意图。
图2为淋巴组织特异性基因表达调控序列的酶切鉴定图。
图3为淋巴组织特异性基因表达调控序列的荧光素酶相对活性图表。
具体实施方式
下述实施例中的方法，如无特别说明，均为常规方法。
马立克氏病(Marek’s disease，MD)是由疱疹病毒科的马立克氏病病毒(Marek’s disease virus，MDV)引起的一种鸡或火鸡的病毒性肿瘤病，致癌毒株主要是MDV-1(血清1型)，可引起鸡各种内脏和组织中以T淋巴细胞肿瘤为特征的增生性疾病，其致病致瘤机制至今未完全明确。MDV-1能编码一种病毒性趋化因子——vIL8，该基因序列与IL8同源，它是α-疱疹病毒中至今发现的唯一与人IL8、鸡IL8(cIL8)等CXC趋化因子同源性较高的病毒因子(virokine)。正如其他疱疹病毒表达的趋化因子，vIL8很可能在病毒感染和/或对宿主免疫调节中起重要作用。本发明首次克隆了vIL8的淋巴细胞特异性正调控序列。
1.vIL8淋巴组织特异性正调控序列的获得
1.1 引物设计及合成
由于启动子区序列复杂，设计两对引物进行目的序列的扩增。引物由上海生工生物工程技术服务有限公司合成。
上游引物P1：5’-CAGACATACTCCTATGCACC，下游引物P2：5’-CCATTTCCAGGCAACAAAT。P1、P2用于扩增vIL8基因的-1362-+81区域，大小为1450bp左右。
上游引物P3：5′CCGCTCGAGGTGTGTAGAGGGCGCATG，下游引物P4：5′CCCAAGCTTCGCCCCAACTGCTCAT。P3、P4用于扩增vIL8基因的-470-+10区域，5’端分别引入XhoI和HindIII酶切位点，大小为500bp左右。
1.2 MDV基因组DNA的提取
MDV GA株为江苏省动物预防医学重点实验室保存。病毒感染次代CEF(鸡胚成纤维细胞)，并维持4d以上，待出现大量的病毒蚀斑时收获。按《分子克隆实验指南(第三版)》提取病毒基因组DNA，溶解于50μL双蒸水中。
1.3 vIL8基因-1362-+81区的PCR扩增
以病毒基因组DNA为模板，以P1、P2为引物，反应体系为50μL，进行PCR扩增vIL8基因启动子区(-1362-+81)片段。反应条件为：94℃ 2min后，94℃ 30s，56℃ 30s，72℃ 90s，反应共25个循环。PCR产物通过电泳(60V，2h)进行分析鉴定。
1.4 vIL8基因-1362-+81区域的克隆及测序
将PCR产物用1％琼脂糖凝胶电泳分离后，试剂盒回收并纯化。将纯化的PCR产物连接到pGEM-Teasy载体(Promega公司产品，货号A1360)，并进行序列测定(由上海生工公司完成)。测序正确的重组质粒命名为pT-PvIL8。
1.5 vIL8的淋巴细胞特异性正调控序列(-470-+10)的序列特征
通过First EF和Dragon Gene StartFinder分析转录起始位点均证明vIL8上游存在转录起始位点且分析结果一致。Promoter 2.0、MatInspector、TESS分析在转录起始位点上游5’非编码区存在多处启动子活性区域。MatInspector、TESS软件分析表明-470-+10区域有多个潜在的转录因子结合元件，包括多个免疫调节相关元件，如5个CRE(cAMP反应元件)，1个NF-κB结合位点，1个Ikaros结合位点。
1.6 vIL8基因淋巴细胞特异性正调控序列(-470-+10)的克隆
以重组质粒pT-PvIL8为模板，以P3、P4为引物，反应体系为50μL，进行PCR扩增。反应条件为：94℃ 2min后，94℃ 30s，58℃ 30s，72℃ 60s，反应共25个循环。PCR产物通过电泳进行分析鉴定。将扩增的PCR片段产物经XhoI和HindIII(宝生物工程(大连)有限公司产品)酶切后电泳回收，与相同酶切电泳回收的pGL3-Basic载体(Promega公司产品，货号为E1741)片段进行连接，转化DH5α后经含氨苄青霉素的LB平板筛选，随机取菌进行小提质粒双酶切鉴定。阳性克隆命名为pGL3-PvIL8。载体构建图见图1。
其核苷酸DNA序列为：
gtgtgtagag ggcgcatgcg tactgccgta agcggtctgc gcatgcgttt tggactgcgc 60
atgcgttttg tgtgtagagg gcgcatgcgt actgcagtaa gtggtctgcg catgcgggtc 120
gtgtgtagag ggcgcatgcg tactgcaaaa tttattgtgg tcctggtttt ctgctatgca 180
ggggtcgtgg gaattttctc atatacattt aagggtcctc atagtcttgt gttccagtat 240
tatcaccttt tgatgcaaac tcctgttcat tttagtgttt cgatgctata gtttatgccc 300
catcgaatgg aaattttcaa taaacaaaat gcatctcacg gtcgtggaat tttaagttgg 360
gggtctccaa tatcacgtgt tggtggagac ccaataacag ggaaatcgcc cgaggcatta 420
cgggtacggt tccatggata tataatgcag ggggtgtggg tttgatgagc agttggggcg 480。
酶切鉴定见图2。在图2中，M列为1kb DNAMarker(上海生工生物工程技术服务有限公司)；1列为pGL3-PvIL8经XhoI和HindIII酶切鉴定。从图2可见在5000bp和500bp附近均有特异条带，分别与pGL3-Basic载体和目的条带大小相符。
1.7 含CMV启动子的pGL3载体的构建
设计1对引物，上游引物P5：5’CCGCTCGAGTTCGCGATGTACG，下游引物P6：5’CCCAAGCTTAATTTCGATAAGCC。分别在5’端引入XhoI和HindIII酶切位点。以P5、P6为引物，以pcDNA3.1/zeo(+)载体(Invitrogen公司产品)为模板扩增CMV启动子序列，反应体系为50μL，反应条件为：94℃ 2min后，94℃ 30s，56℃ 30s，72℃ 90s，反应共25个循环。PCR产物通过电泳(60V，2h)进行分析鉴定。将扩增的CMV启动子片段产物经XhoI和HindIII(宝生物工程(大连)有限公司产品)酶切后电泳回收，与相同酶切电泳回收的pGL3-Basic载体片段进行连接，转化DH5α，并筛选阳性克隆，方法同1.6，阳性克隆命名为pGL3-PCMV。
2 vIL8启动子在淋巴细胞中的特异性活性
2.1 含有vIL8启动子的重组表达载体的转染
鸡胚成纤维细胞(CEF)、鸡胚法氏囊细胞(CEB)分别为9日龄、18日龄SPF鸡胚(购自南京药械厂)按常规方法制备所得，培养基为含5％小牛血清的DMEM(GIBCO/BRL)，用于载体的转染。
转染采用脂质体瞬时转染法进行。脂质体为Invitrogen公司产品，货号是18292-011。转染前1d，次代CEF细胞、原代CEB细胞，均按5×105细胞/孔接种于24孔培养板中，当CEF、CEB细胞生长达80％丰满度时进行转染。每种细胞接种3个孔，分别转染3种质粒，即pGL3-Basic(阴性对照)、pGL3-PvIL8及pGL3-PCMV(阳性对照、内参)。前2种质粒的用量均为0.9μg/孔，pGL3-PCMV为0.1μg/孔(作为内对照)，分别与2μL脂质体混合后进行转染，详细操作见脂质体试剂说明书。重复3次转染。
2.2 启动子对荧光素酶表达物活性的调控效果
转染后72h弃去细胞培养液，PBS洗3次。每孔加入荧光素酶检测试剂盒(Promega产品，货号E1500)内的细胞裂解液100μL。充分裂解细胞后，以12000r/min于4℃离心30min。设定生物化学发光测量仪为延迟6s后计数6s。取20μL上清加入荧光素酶的底物(Luciferase Assay Reagent)100μL检测荧光素酶的活性。为校正转染效率，用所测得的荧光素酶强度值/pGL3-PCMV转染的荧光素酶强度值，得到相对荧光素酶活性，即相对发光值(relative lightunit，RLU)。
如图3所示，vIL8启动子在CEF组中活性接近于0，CEB组活性达到23.2，两者有极显著差异(p<0.01)。同时pGL3-Basic在两种细胞中均基本无荧光素酶表达活性。证明vIL8启动子，即，本发明所述淋巴组织特异性基因表达调控序列(pGL3-PvIL8)在淋巴组织中具有很高的转录活性，具有明显的淋巴细胞特异性功能特征，该启动子在淋巴细胞靶向性表达中可进行相应的应用，特别是在基因治疗淋巴瘤中有一定意义。
经试验还证明以下几种序列也具有正调控功能：
1、有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列片段；
2、遗传变体或缺失型有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列的片段；
3、序列的遗传变体或缺失型有至少150个连续碱基对可衍生自所述启动子-470-+10的DNA序列；
4、序列至少60％与所述启动子-470-+10的DNA序列同源性；
5、序列至少有150个碱基对与所述启动子-470-+10的DNA序列同源性。
gtgtgtagag ggcgcatgcg tactgccgta agcggtctgc gcatgcgttt tggactgcgc 60
atgcgttttg tgtgtagagg gcgcatgcgt actgcagtaa gtggtctgcg catgcgggtc 120
gtgtgtagag ggcgcatgcg tactgcaaaa tttattgtgg tcctggttttc tgctatgca 180
ggggtcgtgg gaattttctc atatacattt aagggtcctc atagtcttgt gttccagtat 240
tatcaccttt tgatgcaaac tcctgttcat tttagtgttt cgatgctata gtttatgccc 300
catcgaatgg aaattttcaa taaacaaaat gcatctcacg gtcgtggaat tttaagttgg 360
gggtctccaa tatcacgtgt tggtggagac ccaataacag ggaaatcgcc cgaggcatta 420
cgggtacggt tccatggata tataatgcag ggggtgtggg tttgatgagc agttggggcg 480
cagacatactcctatgcacc
ccatttccaggcaacaaat
ccgctcgaggtgtgtagagggcgcatg
cccaagcttcgccccaactgctcat
ccgctcgagttcgcgatgtacg
cccaagcttaatttcgataagcc
Claims (1)
1、一种淋巴组织特异性基因表达调控序列，其特征在于具有马立克氏病病毒编码的vIL8基因淋巴组织特异性基因表达正调控的启动子-470-+10的DNA序列，其核苷酸DNA序列为：
gtgtgtagag ggcgcatgcg tactgccgta agcggtctgc gcatgcgttt tggactgcgc 60
atgcgttttg tgtgtagagg gcgcatgcgt actgcagtaa gtggtctgcg catgcgggtc 120
gtgtgtagag ggcgcatgcg tactgcaaaa tttattgtgg tcctggtttt ctgctatgca 180
ggggtcgtgg gaattttctc atatacattt aagggtcctc atagtcttgt gttccagtat 240
tatcaccttt tgatgcaaac tcctgttcat tttagtgttt cgatgctata gtttatgccc 300
catcgaatgg aaattttcaa taaacaaaat gcatctcacg gtcgtggaat tttaagttgg 360
gggtctccaa tatcacgtgt tggtggagac ccaataacag ggaaatcgcc cgaggcatta 420
cgggtacggt tccatggata tataatgcag ggggtgtggg tttgatgagc agttggggcg 480。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996152A (en) * | 1987-12-04 | 1991-02-26 | The United States Of America, As Represented By The Secretary Of Agriculture | Avian herpesvirus amplicon as a eucaryotic expression vector |
| CN1159209A (zh) * | 1995-06-02 | 1997-09-10 | 财团法人化学及血清疗法研究所 | 使用gB基因启动子的重组疱疹病毒 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4996152A (en) * | 1987-12-04 | 1991-02-26 | The United States Of America, As Represented By The Secretary Of Agriculture | Avian herpesvirus amplicon as a eucaryotic expression vector |
| CN1159209A (zh) * | 1995-06-02 | 1997-09-10 | 财团法人化学及血清疗法研究所 | 使用gB基因启动子的重组疱疹病毒 |
Non-Patent Citations (1)
| Title |
|---|
| 马立克氏病病毒类白细胞介素8（vL-8）基因的克隆和表达. 张鑫宇.中国兽医学报，第23卷第6期. 2003 * |
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