【作者】 李鹏；

【导师】 胡明道；

【作者基本信息】 昆明医学院 ， 普通外科学， 2011， 硕士

【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更多还原

【Abstract】 [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.更多还原