重組腺病毒介導(dǎo)恒河猴PD-L1Ig和CD40L胞外區(qū)基因在恒河猴未成熟樹突狀細胞中的表達和鑒定
發(fā)布時間：2018-08-06 13:25
【摘要】：[目的]構(gòu)建和鑒定恒河猴PD-L1Ig和CD40L胞外區(qū)基因重組腺病毒載體。鑒定PD-L1Ig和CD40L胞外區(qū)基因重組腺病毒載體在恒河猴未成熟樹突狀細胞(immature dendritic cell, imDC)中的表達。[方法]1.恒河猴PD-L1Ig和CD40L胞外區(qū)基因的重組腺病毒載體的構(gòu)建、鑒定和包裝。查詢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擴增PD-L11g目的基因。將PD-L1Ig基因雙酶切產(chǎn)物與線性化的GV282載體連接,然后轉(zhuǎn)化感受態(tài)大腸桿菌、氨芐霉素(Amp)篩選轉(zhuǎn)化組菌落、PCR鑒定陽性轉(zhuǎn)化子得到重組質(zhì)粒載體pCMV-3FLAG-IRES-EGFP-PD-L1Ig并對其測序。將pGEM-T-efCD40L質(zhì)粒PCR后的雙酶切產(chǎn)物與線性化重組質(zhì)粒載體pCMV-3FLAG-IRES-EGFP-PD-L1Ig連接,通過轉(zhuǎn)化感受態(tài)大腸桿菌、氨芐霉素(Amp)篩選轉(zhuǎn)化組菌落、PCR鑒定陽性轉(zhuǎn)化子重組穿梭載體pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig并對其測序。重組穿梭載體轉(zhuǎn)染293T細胞,并Western Blot鑒定目的蛋白的表達。應(yīng)用腺病毒AdMax包裝系統(tǒng)法將HEK293細胞和重組載體包裝成重組腺病毒,并用終點稀釋法檢測重組腺病毒滴度。2.分離培養(yǎng)恒河猴imDC,重組腺病毒轉(zhuǎn)染imDC, Western Blot鑒定PD-L1Ig和CD40L胞外區(qū)基因在恒河猴imDC的表達。用猴淋巴細胞分離液提取恒河猴骨髓血中的單個核細胞,免疫磁珠法分選單個核細胞中的CD34+陽性細胞,應(yīng)用細胞因子GM-CSF、IL-4誘導(dǎo)培養(yǎng)其分化為imDC。確定重組腺病毒轉(zhuǎn)染imDC的最佳轉(zhuǎn)染MOI值。轉(zhuǎn)染48小時后提取蛋白并通過Western Blot分別鑒定PD-L1Ig和CD40L基因在imDC中蛋白的表達。[結(jié)果]重組質(zhì)粒載體pCMV-3FLAG-IRES-EGFP-PD-L1Ig序列測序結(jié)果正確。重組穿梭載體pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig序列測序結(jié)果正確。重組穿梭載體轉(zhuǎn)染293T細胞后Western Blot檢測目的融合蛋白,23kD處出現(xiàn)特異性條帶。重組腺病毒滴度為1×10-11 PFU/ml。重組腺病毒轉(zhuǎn)染恒河猴imDC, Western Blot檢測PD-L1Ig,33kD處出現(xiàn)特異性條帶,Western Blot檢測CD40L胞外區(qū),可見29kD處出現(xiàn)特異性條帶。[結(jié)論]成功構(gòu)建pCMV-3FLAG-IRES-EGFP-PD-L1Ig重組質(zhì)粒載體。成功構(gòu)建重組穿梭載體pShuttleCMV-efCD40L-3FLAG-IRES-EGFP-PD-L1Ig。應(yīng)用腺病毒AdMax包裝系統(tǒng)法成功獲得高滴度重組腺病毒。PD-L1Ig和CD40L胞外段基因可以在恒河猴imDC中穩(wěn)定的表達。[展望]重組腺病毒成功轉(zhuǎn)染恒河猴imDC且穩(wěn)定表達,為進一步研究在進行恒河猴肝移植體內(nèi)實驗中PD-L1Ig和CD40L胞外區(qū)基因誘導(dǎo)肝移植免疫耐受提供了理論和實驗基礎(chǔ)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R657.3
本文編號：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R657.3
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相關(guān)期刊論文 前2條
1 許亞明;池詔丞;任明;程龍偉;;樹突狀細胞的研究進展[J];吉林醫(yī)學(xué);2008年13期
2 姚敏學(xué);張黎;支良;胡明道;;重組腺病毒介導(dǎo)恒河猴CD40L胞外段基因在恒河猴未成熟樹突狀細胞中的表達和鑒定[J];現(xiàn)代免疫學(xué);2013年04期
，本文編號：2167869
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