CN1634594A - Mage-1与il-18共表达基因疫苗及其构建方法 - Google Patents
Mage-1与il-18共表达基因疫苗及其构建方法 Download PDFInfo
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Abstract
MAGE-1基因的表达产物是一种肿瘤排斥抗原,该基因在多种恶性肿瘤中高度表达,而在除睾丸外的正常组织中不表达。因此,以MAGE-1为靶点的基因治疗具有广泛的应用前景。IL-18是新近发现的一种具有多种生物功能的细胞因子,是一种良好的免疫佐剂。以pcDNA3为载体,构建出两套表达启动系统,分别表达肿瘤抗原MAGE-1及免疫佐剂IL-18,可有效地增强DNA疫苗的免疫疗效。
Description
技术领域:
本发明属于生物技术领域,特别是涉及一种MAGE-1与IL-18共表达基因疫苗及其构建方法。
背景技术:
基因疫苗又有核酸疫苗、DNA疫苗、基因免疫、DNA免疫等名称和概念。所谓基因疫苗,就是把外源基因克隆到真核质粒表达载体上,然后将重组的质粒DNA直接注射到动物体内,是外源基因在活体内表达,产生的抗原激活机体的免疫系统,引发免疫反应。自从1990年Wolff等首次报道肌肉注射“裸”质粒DNA可被肌肉摄取并表达目的基因多编码的蛋白质以来,基因疫苗受到了广泛关注并获得了迅速的发展。与传统减毒活疫苗和亚单位疫苗相比,基因疫苗有以下优点:①更为有效,基因疫苗可诱导机体产生全面的免疫应答,其保护性免疫应答对不同亚型的病原体具有交叉抵御作用;②安全,无减毒、灭活疫苗可能引起的致病作用,具有可靠的安全性;③能表达经修饰的天然抗原,具有与天然抗原相同的构象和抗原性;④与亚单位疫苗共有的高产性;⑤可将编码不同抗原的基因构建在同一个质粒中,或将不同抗原基因的多种重组质粒联合应用,制备多价基因疫苗;⑥基因疫苗及有预防作用,也为免疫治疗提供强有力的新式武器;⑦生产简便、成本低廉、稳定性好、贮运方便。
MAGE基因是首先从黑色素瘤中发现的一组肿瘤相关抗原基因,至今已发现十余个家族成员,相互之间具有高度同源性。MAGE-1基因在多种不同的肿瘤中都有表达,如食管癌、胃癌、肠癌、肺癌、肝癌、头颈部癌、黑色素瘤、卵巢癌、乳腺癌、肾细胞癌、膀胱癌、骨肉瘤、神经系统肿瘤等等。而在正常组织中除睾丸和胎盘外均不表达。MAGE-1表达的组织分布特点使之成为肿瘤特异性免疫治疗的靶抗原。国内外一些学者对MAGE-1表达产物识别的MHC等位基因限制性进行了深入研究。现已证实MAGE-1全长基因的编码产物可被不同的MHC等位基因编码产物识别、递呈。因此,MAGE-1相关的免疫治疗可不考虑个体的MHC单倍型,具有广泛的应用前景。
IL-18是一种分布广泛的、具有多种生物学活性的细胞因子,它能将NK细胞吞噬细胞介导的非特异性免疫与Th、Tc细胞介导的特异性免疫有机地结合起来,从而在机体的抗肿瘤、抗过敏、抗病原微生物感染等方面发挥重要作用。由于IL-18可诱导IFNγ和GM-CSF的产生,因此,可作为佐剂增强疫苗的免疫效果。
技术内容:
本发明的目的是提供一种可在pcDNA3中共表达肿瘤抗原MAGE-1和免疫佐剂IL-18两种蛋白的MAGE-1与IL-18共表达基因疫苗及其构建方法。
本发明以pcDNA3为真核表达载体,在制备重组表达质粒pcDNA3-IL-18的基础上,设计了一对引物(5’-AAC
AGATCTCGATGTACGGGCCAG-3’和5’-AAG
TCGCGAATGACACCTACTCAG-3’),上游者位于CMV启动子5’端,下游者位于polyA尾3’端。以pcIL-18为模板行PCR。并将PCR产物插入空质粒pcDNA3的BglII及NruI酶切位点之间。
本发明的IL-18共表达基因疫苗载体,其BglII及NruI酶切位点之间核苷酸序列是:
AGATCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT
ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA
ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC
GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGCCAGTACATCT
ACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG
GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT
CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGC
TAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGAAGCTT
ATG TCA GAA GAC TCT TGC GTC AAC TTC AAG GAA ATG ATG TTT ATT GAC AAC
ACG CTT TAC TTT ATA CCT GAA GAA AAT GGA GAC CTG GAA TCA GAC AAC TTT
GGC CGA CTT CAC TGT ACA ACC GCA GTA ATA CGG AAT ATA AAT GAC CAA GTT
CTC TTC GTT GAC AAA AGA CAG CCT GTG TTC GAG GAT ATG ACT GAT ATT GAT
CAA AGT GCC AGT GAA CCC CAG ACC AGA CTG ATA ATA TAC ATG TAC AAA GAC
AGT GAA GAA GTA AGA GGA CTG GCT GTG ACC CTC TCT GTG AAG GAT AGT AAA
ATG TCT ACC CTC TCC TGT AAG AAC AAG ATC ATT TCC TTT GAG GAA ATG GAT
CCA CCT GAA ATA TTG ATG ATA TAC AAA GTG ATC TCA TAT TCT TTC AGA AAC
GTG TTC CAG GAC ACA ACA AGA TGG AGT TTG AAT CTT CAC TGT ATG AAG GAC
ACT TTC TTG CTT GCC AAA AGG AAG ATG ATG CTT TCA AAC TCA TTC TGA
AAAAAAGGATGAAAATGGGGATAAATCTGTAATGTTCACTCTGACTAACTTACATCAAAGTTAGGTGGGGAGGGTTTGT
GTTCCAGAAAGATGATTAGCACACATGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCC
ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAG
GAAATTGCATCGCATTGTCTGAGTAGGTGTAGCGCT
本发明的MAGE-1/IL-18共表达基因疫苗的制备方法是在pcCMV-IL-18的第二表达框中的KpnI和EcoRI位点间可以插入人MAGE-1编码序列,从人肝癌细胞株SMMC-7721总DNA中扩增出MAGE-1序列,再将其插入共表达载体pcCMV-IL-18的多克隆酶切位点中,构建出既表达IL-18又表达MAGE-1的共表达基因疫苗pcIL-18-MAGE。
本发明的MAGE-1/IL-18共表达基因疫苗,其开放读码框为:
gacgagagtc atcatgtctc ttgagcagag gagtctgcac tgcaagcctg aggaagccct 60
tgaggcccaa caagaggccc tgggcctggt gtgtgtgcag gctgccgcct cctcctcctc 120
tcctctggtc ctgggcaccc tggaggaggt gcccactgct gggtcaacag atcctcccca 180
gagtcctcag ggagcctccg cctttcccac taccatcaac ttcactcgac agaggcaacc 240
cagtgagggt tccagcagcc gtgaagagga ggggccaagc acctcttgta tcctggagtc 300
cttgttccga gcagtaatca ctaagaaggt ggctgatttg gttggttttc tgctcctcaa 360
atatcgagcc agggagccag tcacaaaggc agaaatgctg gagagtgtca tcaaaaatta 420
caagcactgt tttcctgaga tcttcggcaa agcctctgag tccttgcagc tggtctttgg 480
cattgacgtg aaggaagcag accccaccgg ccactcctat gtccttgtca cctgcctagg 540
tctctcctat gatggcctgc tgggtgataa tcagatcatg cccaagacag gcttcctgat 600
aattgtcctg gtcatgattg caatggaggg cggccatgct cctgaggagg aaatctggga 660
ggagctgagt gtgatggagg tgtatgatgg gagggagcac agtgcctatg gggagcccag 720
gaagctgctc acccaagatt tggtgcagga aaagtacctg gagtaccggc aggtgccgga 780
cagtgatccc gcacgctatg agttcctgtg gggtccaagg gcccttgctg aaaccagcta 840
tgtgaaagtc cttgagtatg tgatcaaggt cagtgcaaga gttcgctttt tcttcccatc 900
cctgcgtgaa gcagctttga gagaggagga agagggagtc tgagcatgag ttgcagccag 960
ggccagtg 968
本发明的抗原基因MAGE-1两端的酶切位点可以是除了HindIII和ApaI以外的任何位点。
本发明制备出一种生产简便、安全有效、应用广泛、使用方便、价格低廉的共表达基因疫苗。它可特异性的针对在多种肿瘤中高表达的肿瘤抗原MAGE-1其杀伤作用,有由于表达免疫佐剂IL-18而强有力的增强该免疫疗效。
本发明的特色为:
1、具有两个启动子(CMV),即两套表达系统;
2、可同时表达抗原基因及免疫佐剂IL-18。其优点是免疫佐剂可有效地增强抗原基因诱导的特异性免疫应答。
3、pcCMV-IL-18只有一处多克隆酶切位点,可以有针对性的进行插入目的基因。该载体中具有两套表达启动系统,其中一套多克隆酶切位点已完全被IL-18所替代,因此仅余另一套多克隆酶切位点。所以,除第一个(HindIII)和最后一个(ApaI)酶切位点,其余酶切位点均可作为插入目的基因时的备选方案。
该共表达载体(pcCMV-IL-18)若需装载另外的2个目的基因,则只需将其中一个目的基因取代IL-18,而另一个目的基因也如同我们的MAGE-1基因一样有多个备选的酶切位点供参考。
附图说明:
图1是本发明所使用的pcDNA3结构图谱;
图2是本发明构建的pcIL-18表达质粒的结构图谱;
图3是本发明共表达基因疫苗载体pcCMV-IL-18结构图谱;
图4是本发明共表达基因疫苗pcIL-18-MAGE结构图谱。
具体实施方式:
1.IL-18重组质粒pcIL-18的构建
1.1小鼠肝组织总RNA的提取:Balb/C小鼠,雌性,19-22天。
取肝脏50-100mg,按照Trizol Reagent说明提取肝组织的总RNA,-70℃保存。
1.2mIL-18特异性引物设计:依据引物设计原则,参照Genbank中mIL-18的mRNA序列(NM 008360)设计一对引物,P1和P2。并在其5‘端分别引入HindIII和ApaI酶切位点。送交公司合成。
P1:5’-GG
AAGCTTATGGCTGCCATGTCAGAAGACTC-3’;
P2:5’-ATA
GGGCCCAGGCGCATGTGTGCTAATCATC-3’。
1.3逆转录合成cDNA:参照AMV Reverse transcriptase说明方法,取小鼠肝组织总RNA 5μl,Oligo(dT)2μl,DEPC H2O 13μl,混合,70℃孵育5min,冰水冷却5min。之后加入dNTP Mixture5μl,AMV 5×Buffer 10μl,Rnasin 2μl,AMV Reversetranscriptase 3μl,补水至总体积为50μl,混匀,42℃孵育90min,95℃灭活5min。
1.4PCR扩增mIL-18:0.2ml离心管中加入10×Buffer 5μl,Mg2+5μl,dNTP 4μl,P1、P2各1μl,cDNA 10μl,Taq酶1μl,补足水至50μl反应体系,再加入液体石蜡25μl。对照中将P1、P2换成β-actin引物。反应条件:94℃,45sec,55℃,1min,72℃,1min,共40个循环。之后72℃充分延伸7min。1%琼脂糖凝胶电泳,用试剂盒将目的条带回收。
1.5构建重组质粒pcIL-18:将mIL-18RT-PCR产物及载体pcDNA3分别做HindIII和ApaI双酶切,再将酶切产物用T4 Ligase 16℃过夜连接。参照《分子克隆实验指南》,将连接产物转化大肠杆菌DH5α。用含有氨苄的培养板筛选出pcIL-18重组质粒。按照日常型质粒DNA小量纯化试剂盒说明提取重组质粒pcIL-18。
1.6重组质粒的鉴定:
1.6.1酶切鉴定:用HindIII和ApaI做双酶切鉴定,同时以空质粒pcDNA3为对照。1%琼脂糖凝胶电泳判断结果。
1.6.2测序分析:将重组质粒送交测序公司测序,将测序结果与Genbank进行比较分析。
2含mIL-18共表达系统的构建
2.1引物设计:依据引物设计原则,参照pcDNA3图谱设计一对引物,P3和P4。并在其5‘端分别引入BglII和NruI酶切位点。送交公司合成。
P3:5’-AAC
AGATCTCGATGTACGGGCCAG-3’
P4:5’-AAG
TCGCGAATGACACCTACTCAG-3’
2.2以pcIL-18为模板进行PCR,扩增CMV-IL-18-polyA序列:
取pcIL-18质粒1μl,taqE 2μl,10×Buffer 5μl,引物各1μl,dNTP5μl,Mg+5μl,补水至50μl。循环程序为:94℃预变性5min;94℃ 1min,55℃ 1min,72℃ 2min,30个循环;72℃充分延伸10min。将PCR产物在1%琼脂糖凝胶作电泳,并将特异条带用试剂盒作凝胶DNA回收。
2.3以pcDNA3为载体,构建含mIL-18的共表达系统:将CMV-IL-18-polyA的PCR产物与空质粒pcDNA3分别作BglII和NruI双酶切,回收酶切产物,用T4 Ligase 16℃过夜连接。将连接产物转化大肠杆菌DH5α。用含有氨苄的培养板筛选出重组质粒pcCMV-IL-18。
3MAGE-1与mIL-18共表达系统的构建
3.1SMMC-7721总DNA的提取:取1×107细胞,用试剂盒提取细胞总DNA。
3.2MAGE-1特异性引物的设计:根据引物设计原则,参照Genbank中MAGE-1的编码序列(AY148486),设计一对引物P5和P6,并在其上下游分别加入KpnI和EcoRI。由公司合成。
P5:5’-ATTGGTACCGACGAGAGTCATCATGTC-3’
P6:5’-TTAGAATTCCTGGAAGGTGCACTGGCC-3’
3.3从SMMC-7721总DNA中扩增MAGE-1全长序列:取DNA1μl,taqE 2μl,10×Buffer 5μl,引物各1μl,dNTP5μl,Mg+5μl,补水至50μl。循环程序为:94℃预变性5min;94℃1min,55℃1min,72℃1.5min,40个循环;72℃充分延伸10min。将PCR产物在1%琼脂糖凝胶作电泳,并将特异条带用试剂盒作凝胶DNA回收。
3.4构建pcIL-18-MAGE共表达质粒:将MAGE-1的PCR产物与含mIL-18的共表达质粒pcCMV-IL-18分别作KpnI和EcoRI双酶切,回收酶切产物,用T4 Ligase 16℃过夜连接。构建出pcIL-18-MAGE。
4pcIL-18-MAGE的大量生产
4.1制备感受态大肠杆菌DH5α(参见《分子克隆实验指南》)
4.2用pcIL-18-MAGE共表达质粒与MAGE-1的连接产物pcIL-18-MAGE转化DH5α。并用含有氨苄的培养板筛选。
4.3挑取单菌落进行KpnI和EcoRI双酶切鉴定。
4.4用无内毒素型质粒大量提取试剂盒按照说明提取质粒。
本文中标准缩略语的中文释义
MAGE-1:黑色素瘤相关抗原
IL-18:白细胞介素-18
PcDNA3:真核表达质粒
CMV:巨细胞病毒
MHC:主要组织相容性复合体
Th、Tc、NK:辅助性T细胞、细胞毒性T细胞、天然杀伤细胞
AMV Reverse transcriptase:禽病毒逆转录酶
dNTP mixture:脱氧核糖核苷酸混合物
CDNA:互补DNA
T4 ligase:T4连接酶
Claims (5)
1、一种IL-18共表达基因载体pcCMV-18的构建方法,其特征在于:以pcDNA3为真核表达载体,在制备重组表达质粒pcDNA3-IL-18的基础上,设计了一对引物(5’-AAC
AGATCTCGATGTACGGGCCAG-3’和5’-AAG
TCGCGAATGACACCTACTCAG-3’),上游者位于CMV启动子5’端,下游者位于polyA尾3’端。以pcIL-18为模板行PCR。并将PCR产物插入空质粒pcDNA3的BglII及NruI酶切位点之间。
2、一种IL-18共表达基因疫苗载体,其BglII及NruI酶切位点之间核苷酸序列是:
AGATCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT
ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA
ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC
GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCT
ACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG
GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGT
CGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGC
TAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGAAGCTT
ATG TCA GAA GAC TCT TGC GTC AAC TTC AAG GAA ATG ATG TTT ATT GAC AAC
ACG CTT TAC TTT ATA CCT GAA GAA AAT GGA GAC CTG GAA TCA GAC AAC TTT
GGC CGA CTT CAC TGT ACA ACC GCA GTA ATA CGG AAT ATA AAT GAC CAA GTT
CTC TTC GTT GAC AAA AGA CAG CCT GTG TTC GAG GAT ATG ACT GAT ATT GAT
CAA AGT GCC AGT GAA CCC CAG ACC AGA CTG ATA ATA TAC ATG TAC AAA GAC
AGT GAA GAA GTA AGA GGA CTG GCT GTG ACC CTC TCT GTG AAG GAT AGT AAA
ATG TCT ACC CTC TCC TGT AAG AAC AAG ATC ATT TCC TTT GAG GAA ATG GAT
CCA CCT GAA ATA TTG ATG ATA TAC AAA GTG ATC TCA TAT TCT TTC AGA AAC
GTG TTC CAG GAC ACA ACA AGA TGG AGT TTG AAT CTT CAC TGT ATG AAG GAC
ACT TTC TTG CTT GCC AAA AGG AAG ATG ATG CTT TCA AAC TCA TTC TGA
AAAAAAGGATGAAAATGGGGATAAATCTGTAATGTTCACTCTGACTAACTTACATCAAAGTTAGGTGGGGAGGGTTTGT
GTTCCAGAAAGATGATTAGCACACATGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCC
ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAG
GAAATTGCATCGCATTGTCTGAGTAGGTGTAGCGCT
3、一种MAGE-1/IL-18共表达基因疫苗的制备方法,其特征在于:在pcCMV-IL-18的第二表达框中的KpnI和EcoRI位点间可以插入人MAGE-1编码序列,从人肝癌细胞株SMMC-7721总DNA中扩增出MAGE-1序列,再将其插入共表达载体pcCMV-IL-18的多克隆酶切位点中,构建出既表达IL-18又表达MAGE-1的共表达基因疫苗pcIL-18-MAGE。
4、一种MAGE-1/IL-18共表达基因疫苗,其开放读码框为:
gacgagagtc atcatgtctc ttgagcagag gagtctgcac tgcaagcctg aggaagccct 60
tgaggcccaa caagaggccc tgggcctggt gtgtgtgcag gctgccgcct cctcctcctc 120
tcctctggtc ctgggcaccc tggaggaggt gcccactgct gggtcaacag atcctcccca 180
gagtcctcag ggagcctccg cctttcccac taccatcaac ttcactcgac agaggcaacc 240
cagtgagggt tccagcagcc gtgaagagga ggggccaagc acctcttgta tcctggagtc 300
cttgttccga gcagtaatca ctaagaaggt ggctgatttg gttggttttc tgctcctcaa 360
atatcgagcc agggagccag tcacaaaggc agaaatgctg gagagtgtca tcaaaaatta 420
caagcactgt tttcctgaga tcttcggcaa agcctctgag tccttgcagc tggtctttgg 480
cattgacgtg aaggaagcag accccaccgg ccactcctat gtccttgtca cctgcctagg 540
tctctcctat gatggcctgc tgggtgataa tcagatcatg cccaagacag gcttcctgat 600
aattgtcctg gtcatgattg caatggaggg cggccatgct cctgaggagg aaatctggga 660
ggagctgagt gtgatggagg tgtatgatgg gagggagcac agtgcctatg gggagcccag 720
gaagctgctc acccaagatt tggtgcagga aaagtacctg gagtaccggc aggtgccgga 780
cagtgatccc gcacgctatg agttcctgtg gggtccaagg gcccttgctg aaaccagcta 840
tgtgaaagtc cttgagtatg tgatcaaggt cagtgcaaga gttcgctttt tcttcccatc 900
cctgcgtgaa gcagctttga gagaggagga agagggagtc tgagcatgag ttgcagccag 960
ggccagtg 968
5、根据权利要求3所述的MAGE-1/IL-18共表达基因疫苗的制备方法,其特征在于:抗原基因MAGE-1两端的酶切位点可以是除了HindIII和ApaI以外的任何位点。
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