恒河猴共刺激分子PD-L1基因胞外区与IgG_1Fc融合基因腺病毒载体的构建与表达
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摘要
[目的]构建恒河猴共刺激分子PD-L1蛋白基因胞外区与IgG,Fc段的融合基因腺病毒载体,转染293细胞包装并表达PD-L1Ig融合蛋白。
[方法] (一)在Genebank中找到恒河猴目的基因PD-L1和IgG1Fc的序列,根据对基因结构的说明确定PD-L1和IgG1Fc胞外区序列。以目的基因的序列为基础设计59个碱基的引物,引物间两两重叠21个碱基,采用重叠延伸PCR法合成该目的基因。
(二)合成的目的基因与pGEM-T连接,转化感受态E.coli TOP10,克隆恒河猴PD-L1胞外区基因和IgG1Fc段基因。
(三)以salⅠ和EcoV双酶切pGEM-T/IgG1Fc、pGEM-T/PD-L1得到PD-L1基因和pGEM-T/IgG1Fc线性化载体,体外连接转化感受态E.coli TOP10,克隆pGEM-T/PD-L1Ig。
(四)将PD-L1Ig基因和sal I、EcoV双酶切的pShuttle-CMV线性化载体体外连接并转化感受态E.coli TOP 10,克隆pShuttle-CMV/PD-LlIg重组穿梭质粒。
(五)将pAdEasy-1和线性化的pShuttle-CMV/PD-LlIg共电转至BJ5183细胞中同源重组,构建重组腺病毒载体pAdEasy-1/PD-L1Ig
(六)以脂质体2000将pAdEasy-1/PD-L1Ig转染至AD293细胞中,经培养将获得的重组腺病毒原液反复感染AD293细胞并经多轮扩增和纯化。
(七)RT-PCR和western bloting检测目的基因的表达。
[结果]克隆的PD-L1胞外区基因和IgG1Fc大小分别为660bp和280bp,经测序与Genebank中两个基因大小和序列相符。在pGEM-T上构建了PD-L1Ig融合基因,PCR和酶切鉴定可见PD-L1Ig基因的目的条带,大小为940bp,测序正确。构建的重组腺病毒载体经酶切和PCR鉴定均正确。重组腺病毒载体转染293细胞后,经过多轮扩增和纯化得到了滴度为6.5×109pfu/ml重组腺病毒。RT-PCR可见条带大小为940bp,与目的基因大小相符。western bloting可见约35.1KDa的条带,与目的蛋白PD-L1Ig的大小相符。
[结论]成功构建了pAdEasy-1/PD-L1Ig重组腺病毒载体,得到了能够表达共刺激分子PD-L1Ig融合蛋白的高滴度重组腺病毒
[展望]pAdEasy-1/PD-L1Ig重组腺病毒在后续的实验中将被转染至恒河猴未成熟树突状细胞,使其表达PD-L1Ig,激活PD-1/PDL-1共刺激通路,预计可有效诱导恒河猴肝移植免疫耐受
[Object] To construct fusion gene of PD-L1ECD and IgG1Fc in recombinant adenoviral vector in BJ5183 which can express Rhesus fusion protein of PD-L1ECD and IgG1Fc.
[Methods] (1) To discover the Rhesus's sequences of PD-L1ECD and IgG1Fc in Genebank.To design some 59bp primers which overlap 21bp on the basis of the sequences of PD-L1ECD and IgG1Fc according to the Genebank's gaidline to this two genes. To synthesize the gene of PD-L1ECD and IgG1Fc through overlap extension PCR.
(2) The synthesized genes of PD-L1ECD and IgG1Fc are ligased to pGEM-T in vitro and transformed to E.coliTOP10 to clone the aimed genes.
(3) Linearized pGEM-T/IgG1Fc and PD-L1 ECD gene which are doubly digested by salⅠand EcoⅤrespectively are ligased and transformed to E.coliTOP10 to clone the fusion gene pGEM-T/PD-L1Ig.
(4) Linearized pGEM-T/IgG1Fc which is doubly digested by salⅠand EcoⅤand PD-L1 ECD gene are ligased and transformed to E.coliTOP10 to clone the pShuttle-CMV/PD-L1Ig.
(5) Linearized pShuttle-CMV/PD-L1Ig and pAdEasy-1 are co-transformed to BJ5183 by electroporation through homologous recombination to clone the pAdEasy-1/PD-L1Ig.
(6) Linearized pAdEasy-1/PD-L1Ig is transformed by Lipofectamin2000 to AD293 which is cultured to be original adenoviral fruid.The original adenoviral fruid are infected to the new AD293 that are amplified and purified repeatedly.
(7)RT-PCR and Western Bloting testify what the recombinant adenovirus can express.
[Result] PD-L1ECD and IgG1Fc were cloned out whose length are 660bp and 280bp respectively in correspondence with the length and sequence in Genebank. PD-L1Ig fusion gene whose length sequence are testified by restriction digestion、PCR and sequencing was constructed in pGEM-T. Recombinant Ad plasmid was testified accurate by restriction digestion and PCR.The titer of Recombinant Ad was measured as 6.5×109pfu/ml after it was transfected to 293 cells and amplified and purified repeatedly.RT-PCR display the band of 940bp matched with the length of PD-L1Ig cDNA.Western bloting display 35.1KDa of band matched with PD-L1Ig protein size.
[Conclusion] Constructed the high titer recombinant adenovirus successfully which can express costimulatory molecule PD-L1Ig fusion protein.
[Prospect] Recombinant adenovirus which can express PD-L1Ig of costimulatory molecule will be transfected to iDC which could activiate PD-1/PD-L1 pathway that would be predicted to induce immunological tolerance of Rhesus liver transplantation effectively in the continued experimental research.
[方法] (一)在Genebank中找到恒河猴目的基因PD-L1和IgG1Fc的序列,根据对基因结构的说明确定PD-L1和IgG1Fc胞外区序列。以目的基因的序列为基础设计59个碱基的引物,引物间两两重叠21个碱基,采用重叠延伸PCR法合成该目的基因。
(二)合成的目的基因与pGEM-T连接,转化感受态E.coli TOP10,克隆恒河猴PD-L1胞外区基因和IgG1Fc段基因。
(三)以salⅠ和EcoV双酶切pGEM-T/IgG1Fc、pGEM-T/PD-L1得到PD-L1基因和pGEM-T/IgG1Fc线性化载体,体外连接转化感受态E.coli TOP10,克隆pGEM-T/PD-L1Ig。
(四)将PD-L1Ig基因和sal I、EcoV双酶切的pShuttle-CMV线性化载体体外连接并转化感受态E.coli TOP 10,克隆pShuttle-CMV/PD-LlIg重组穿梭质粒。
(五)将pAdEasy-1和线性化的pShuttle-CMV/PD-LlIg共电转至BJ5183细胞中同源重组,构建重组腺病毒载体pAdEasy-1/PD-L1Ig
(六)以脂质体2000将pAdEasy-1/PD-L1Ig转染至AD293细胞中,经培养将获得的重组腺病毒原液反复感染AD293细胞并经多轮扩增和纯化。
(七)RT-PCR和western bloting检测目的基因的表达。
[结果]克隆的PD-L1胞外区基因和IgG1Fc大小分别为660bp和280bp,经测序与Genebank中两个基因大小和序列相符。在pGEM-T上构建了PD-L1Ig融合基因,PCR和酶切鉴定可见PD-L1Ig基因的目的条带,大小为940bp,测序正确。构建的重组腺病毒载体经酶切和PCR鉴定均正确。重组腺病毒载体转染293细胞后,经过多轮扩增和纯化得到了滴度为6.5×109pfu/ml重组腺病毒。RT-PCR可见条带大小为940bp,与目的基因大小相符。western bloting可见约35.1KDa的条带,与目的蛋白PD-L1Ig的大小相符。
[结论]成功构建了pAdEasy-1/PD-L1Ig重组腺病毒载体,得到了能够表达共刺激分子PD-L1Ig融合蛋白的高滴度重组腺病毒
[展望]pAdEasy-1/PD-L1Ig重组腺病毒在后续的实验中将被转染至恒河猴未成熟树突状细胞,使其表达PD-L1Ig,激活PD-1/PDL-1共刺激通路,预计可有效诱导恒河猴肝移植免疫耐受
[Object] To construct fusion gene of PD-L1ECD and IgG1Fc in recombinant adenoviral vector in BJ5183 which can express Rhesus fusion protein of PD-L1ECD and IgG1Fc.
[Methods] (1) To discover the Rhesus's sequences of PD-L1ECD and IgG1Fc in Genebank.To design some 59bp primers which overlap 21bp on the basis of the sequences of PD-L1ECD and IgG1Fc according to the Genebank's gaidline to this two genes. To synthesize the gene of PD-L1ECD and IgG1Fc through overlap extension PCR.
(2) The synthesized genes of PD-L1ECD and IgG1Fc are ligased to pGEM-T in vitro and transformed to E.coliTOP10 to clone the aimed genes.
(3) Linearized pGEM-T/IgG1Fc and PD-L1 ECD gene which are doubly digested by salⅠand EcoⅤrespectively are ligased and transformed to E.coliTOP10 to clone the fusion gene pGEM-T/PD-L1Ig.
(4) Linearized pGEM-T/IgG1Fc which is doubly digested by salⅠand EcoⅤand PD-L1 ECD gene are ligased and transformed to E.coliTOP10 to clone the pShuttle-CMV/PD-L1Ig.
(5) Linearized pShuttle-CMV/PD-L1Ig and pAdEasy-1 are co-transformed to BJ5183 by electroporation through homologous recombination to clone the pAdEasy-1/PD-L1Ig.
(6) Linearized pAdEasy-1/PD-L1Ig is transformed by Lipofectamin2000 to AD293 which is cultured to be original adenoviral fruid.The original adenoviral fruid are infected to the new AD293 that are amplified and purified repeatedly.
(7)RT-PCR and Western Bloting testify what the recombinant adenovirus can express.
[Result] PD-L1ECD and IgG1Fc were cloned out whose length are 660bp and 280bp respectively in correspondence with the length and sequence in Genebank. PD-L1Ig fusion gene whose length sequence are testified by restriction digestion、PCR and sequencing was constructed in pGEM-T. Recombinant Ad plasmid was testified accurate by restriction digestion and PCR.The titer of Recombinant Ad was measured as 6.5×109pfu/ml after it was transfected to 293 cells and amplified and purified repeatedly.RT-PCR display the band of 940bp matched with the length of PD-L1Ig cDNA.Western bloting display 35.1KDa of band matched with PD-L1Ig protein size.
[Conclusion] Constructed the high titer recombinant adenovirus successfully which can express costimulatory molecule PD-L1Ig fusion protein.
[Prospect] Recombinant adenovirus which can express PD-L1Ig of costimulatory molecule will be transfected to iDC which could activiate PD-1/PD-L1 pathway that would be predicted to induce immunological tolerance of Rhesus liver transplantation effectively in the continued experimental research.
引文
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2曹培丽,李媛,陈超等猪肺炎霉形体p52基因重组腺病毒的构建及其免疫特性中国兽医科学[J]2009,39(08)：712-717
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4胡朝全,孙诚谊,孙连生等重组腺病毒载体介导人抑瘤素M基因对胰腺癌细胞体外增殖的抑制作用[J]中国现代医学杂志2008 18(4)1060-1062
5王长胜林嘉华吴朝阳重组腺病毒载体A d 5-hBDNF-EGFP的构建与鉴定中国组织工程研究与临床康复[J]2009 13(20)3947-3951
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8杨月峰吴祖泽王立生重组腺病毒介导的前列腺癌基因治疗研究进展中国药理学与毒理学杂志[J]2009 23(4)316-319
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11张耀文王芹李进等重组腺病毒Ad-Rb94基因联合放疗对人肝癌细胞生长的抑制作用中国辐射卫生[J]2008(17)2：147-149
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