重组腺病毒介导恒河猴PD-L1Ig和CD40L胞外区基因在恒河猴未成熟树突状细胞中的表达和鉴定
发布时间：2018-08-06 13:25
【摘要】：[目的]构建和鉴定恒河猴PD-L1Ig和CD40L胞外区基因重组腺病毒载体。鉴定PD-L1Ig和CD40L胞外区基因重组腺病毒载体在恒河猴未成熟树突状细胞(immature dendritic cell, imDC)中的表达。[方法]1.恒河猴PD-L1Ig和CD40L胞外区基因的重组腺病毒载体的构建、鉴定和包装。查询GeneBank中恒河猴PD-L1和IgGlFc基因序列并基因合成PD-L1Ig目的基因序列,然后PCR扩增PD-L11g目的基因。将PD-L1Ig基因双酶切产物与线性化的GV282载体连接,然后转化感受态大肠杆菌、氨苄霉素(Amp)筛选转化组菌落、PCR鉴定阳性转化子得到重组质粒载体pCMV-3FLAG-IRES-EGFP-PD-L1Ig并对其测序。将pGEM-T-efCD40L质粒PCR后的双酶切产物与线性化重组质粒载体pCMV-3FLAG-IRES-EGFP-PD-L1Ig连接,通过转化感受态大肠杆菌、氨苄霉素(Amp)筛选转化组菌落、PCR鉴定阳性转化子重组穿梭载体pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig并对其测序。重组穿梭载体转染293T细胞,并Western Blot鉴定目的蛋白的表达。应用腺病毒AdMax包装系统法将HEK293细胞和重组载体包装成重组腺病毒,并用终点稀释法检测重组腺病毒滴度。2.分离培养恒河猴imDC,重组腺病毒转染imDC, Western Blot鉴定PD-L1Ig和CD40L胞外区基因在恒河猴imDC的表达。用猴淋巴细胞分离液提取恒河猴骨髓血中的单个核细胞,免疫磁珠法分选单个核细胞中的CD34+阳性细胞,应用细胞因子GM-CSF、IL-4诱导培养其分化为imDC。确定重组腺病毒转染imDC的最佳转染MOI值。转染48小时后提取蛋白并通过Western Blot分别鉴定PD-L1Ig和CD40L基因在imDC中蛋白的表达。[结果]重组质粒载体pCMV-3FLAG-IRES-EGFP-PD-L1Ig序列测序结果正确。重组穿梭载体pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig序列测序结果正确。重组穿梭载体转染293T细胞后Western Blot检测目的融合蛋白,23kD处出现特异性条带。重组腺病毒滴度为1×10-11 PFU/ml。重组腺病毒转染恒河猴imDC, Western Blot检测PD-L1Ig,33kD处出现特异性条带,Western Blot检测CD40L胞外区,可见29kD处出现特异性条带。[结论]成功构建pCMV-3FLAG-IRES-EGFP-PD-L1Ig重组质粒载体。成功构建重组穿梭载体pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig。应用腺病毒AdMax包装系统法成功获得高滴度重组腺病毒。PD-L1Ig和CD40L胞外段基因可以在恒河猴imDC中稳定的表达。[展望]重组腺病毒成功转染恒河猴imDC且稳定表达,为进一步研究在进行恒河猴肝移植体内实验中PD-L1Ig和CD40L胞外区基因诱导肝移植免疫耐受提供了理论和实验基础。
[Abstract]:[objective] to construct and identify the recombinant adenovirus vector of rhesus monkey PD-L1Ig and CD40L. The expression of recombinant adenovirus vector of PD-L1Ig and CD40L in immature dendritic cells (immature dendritic cell, imDC) of rhesus monkey was identified. [methods] 1. Construction, Identification and Packaging of Recombinant adenovirus Vector of PD-L1Ig and CD40L extracellular region genes in Rhesus Monkey. The PD-L1 and IgGlFc genes of rhesus monkey in GeneBank were sequenced and the PD-L1Ig target gene sequence was synthesized. Then the PD-L11g target gene was amplified by PCR. The double enzyme digested product of PD-L1Ig gene was ligated with the linearized GV282 vector, then transformed into the competent Escherichia coli. The recombinant plasmid pCMV-3FLAG-IRES-EGFP-PD-L1Ig was obtained and sequenced by screening the positive transformants of ampicillin (Amp). The double enzyme digested product of pGEM-T-efCD40L plasmid PCR was ligated with the linearized recombinant plasmid vector pCMV-3FLAG-IRES-EGFP-PD-L1Ig. The recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig was identified and sequenced by transformation of Escherichia coli and ampicillin (Amp). The recombinant shuttle vector was transfected into 293T cells and the expression of target protein was identified by Western Blot. Adenovirus AdMax packaging system was used to package HEK293 cells and recombinant vectors into recombinant adenovirus, and the titer of recombinant adenovirus was detected by end-point dilution method. Rhesus monkey imDCwas isolated and cultured. The recombinant adenovirus was transfected into imDC, Western Blot to identify the expression of PD-L1Ig and CD40L extracellular domain genes in imDC of rhesus monkey. The mononuclear cells from rhesus monkey bone marrow blood were extracted from monkey lymphocyte isolate and the CD34 positive cells were isolated by immunomagnetic beads. The mononuclear cells were induced to differentiate into imDCby the cytokine GM-CSF- IL-4. The optimal MOI value of imDC transfection by recombinant adenovirus was determined. The protein was extracted 48 hours after transfection and the expression of PD-L1Ig and CD40L genes in imDC was identified by Western Blot. [results] the sequence of recombinant plasmid pCMV-3FLAG-IRES-EGFP-PD-L1Ig was correct. The sequence of recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig was correct. After the recombinant shuttle vector was transfected into 293T cells, the specific band of fusion protein 23 KD was detected by Western Blot. The recombinant adenovirus titer was 1 脳 10-11 PFU / ml. The recombinant adenovirus transfected rhesus monkey imDC, Western Blot showed a specific band at 33 KD of PD-L1 IgA. Western Blot was used to detect the extracellular region of CD40L, and a specific band was found in 29kD. [conclusion] pCMV-3FLAG-IRES-EGFP-PD-L1Ig recombinant plasmid was successfully constructed. The recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig. High titer recombinant adenovirus. PD-L1Ig and CD40L extracellular segment genes were successfully expressed in rhesus monkey imDC by adenovirus AdMax packaging system. [prospect] Recombinant adenovirus was successfully transfected into rhesus monkey imDC and expressed stably, which provides a theoretical and experimental basis for further study on the induction of immune tolerance by PD-L1Ig and CD40L extracellular region genes in rhesus monkey liver transplantation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3
本文编号：2167869
[Abstract]:[objective] to construct and identify the recombinant adenovirus vector of rhesus monkey PD-L1Ig and CD40L. The expression of recombinant adenovirus vector of PD-L1Ig and CD40L in immature dendritic cells (immature dendritic cell, imDC) of rhesus monkey was identified. [methods] 1. Construction, Identification and Packaging of Recombinant adenovirus Vector of PD-L1Ig and CD40L extracellular region genes in Rhesus Monkey. The PD-L1 and IgGlFc genes of rhesus monkey in GeneBank were sequenced and the PD-L1Ig target gene sequence was synthesized. Then the PD-L11g target gene was amplified by PCR. The double enzyme digested product of PD-L1Ig gene was ligated with the linearized GV282 vector, then transformed into the competent Escherichia coli. The recombinant plasmid pCMV-3FLAG-IRES-EGFP-PD-L1Ig was obtained and sequenced by screening the positive transformants of ampicillin (Amp). The double enzyme digested product of pGEM-T-efCD40L plasmid PCR was ligated with the linearized recombinant plasmid vector pCMV-3FLAG-IRES-EGFP-PD-L1Ig. The recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig was identified and sequenced by transformation of Escherichia coli and ampicillin (Amp). The recombinant shuttle vector was transfected into 293T cells and the expression of target protein was identified by Western Blot. Adenovirus AdMax packaging system was used to package HEK293 cells and recombinant vectors into recombinant adenovirus, and the titer of recombinant adenovirus was detected by end-point dilution method. Rhesus monkey imDCwas isolated and cultured. The recombinant adenovirus was transfected into imDC, Western Blot to identify the expression of PD-L1Ig and CD40L extracellular domain genes in imDC of rhesus monkey. The mononuclear cells from rhesus monkey bone marrow blood were extracted from monkey lymphocyte isolate and the CD34 positive cells were isolated by immunomagnetic beads. The mononuclear cells were induced to differentiate into imDCby the cytokine GM-CSF- IL-4. The optimal MOI value of imDC transfection by recombinant adenovirus was determined. The protein was extracted 48 hours after transfection and the expression of PD-L1Ig and CD40L genes in imDC was identified by Western Blot. [results] the sequence of recombinant plasmid pCMV-3FLAG-IRES-EGFP-PD-L1Ig was correct. The sequence of recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig was correct. After the recombinant shuttle vector was transfected into 293T cells, the specific band of fusion protein 23 KD was detected by Western Blot. The recombinant adenovirus titer was 1 脳 10-11 PFU / ml. The recombinant adenovirus transfected rhesus monkey imDC, Western Blot showed a specific band at 33 KD of PD-L1 IgA. Western Blot was used to detect the extracellular region of CD40L, and a specific band was found in 29kD. [conclusion] pCMV-3FLAG-IRES-EGFP-PD-L1Ig recombinant plasmid was successfully constructed. The recombinant shuttle vector pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig. High titer recombinant adenovirus. PD-L1Ig and CD40L extracellular segment genes were successfully expressed in rhesus monkey imDC by adenovirus AdMax packaging system. [prospect] Recombinant adenovirus was successfully transfected into rhesus monkey imDC and expressed stably, which provides a theoretical and experimental basis for further study on the induction of immune tolerance by PD-L1Ig and CD40L extracellular region genes in rhesus monkey liver transplantation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3
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相关期刊论文 前2条
1 许亚明;池诏丞;任明;程龙伟;;树突状细胞的研究进展[J];吉林医学;2008年13期
2 姚敏学;张黎;支良;胡明道;;重组腺病毒介导恒河猴CD40L胞外段基因在恒河猴未成熟树突状细胞中的表达和鉴定[J];现代免疫学;2013年04期
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