本文選題：TLR4 + MD2��； 參考：《寧夏醫(yī)科大學》2012年碩士論文

【摘要】：目的采用基因工程和分子生物學的方法，構建體外表達Toll樣受體及與熱原反應相關基因的細胞學模型，為檢測注射劑的熱原反應提供實驗室使用工具。 方法克隆人Toll樣受體4（TLR4）和髓樣分化蛋白（MD2），構建pcDNA.3.1/myc-His(-)B-TLR4、pcDNA.3.1/myc-His(-)B-MD2真核表達載體。 ①pMD19-T-TLR4、pMD19-T-MD2克隆載體的構建及鑒定：提取人外周血單核細胞總RNA，利用RT-PCR得到TLR4-1、TLR4-2和MD2的DNA�；厥誔CR產(chǎn)物連接于pMD-19T載體上進行克隆和測序。②pcDNA.3.1/myc-His(-)B-TLR4表達載體的構建及鑒定：測序鑒定后的pMD19-T-TLR4-2載體質粒與pcDNA.3.1/myc-His(-)B表達載體質粒同時進行EcoRI和XhoI雙酶切，經(jīng)連接酶作用后，轉化至感受態(tài)E.coliTop10，氨芐青霉素篩選，挑取陽性克隆并酶切鑒定，得pcDNA.3.1/myc-His(-)B-TLR4-2載體質粒。鑒定合格的pcDNA.3.1/myc-His(-)B-TLR4-2載體質粒與pMD19-T-TLR4-1載體質粒同時進行hpaI和BamHI雙酶切，經(jīng)連接酶作用后，轉化至感受態(tài)E.coliTop10，氨芐青霉素篩選，挑取陽性克隆并酶切鑒定，最終獲得pcDNA.3.1/myc-His(-)B-TLR4載體質粒。③pcDNA.3.1/myc-His(-)B-MD2表達載體的構建及鑒定：pMD19-T-MD2載體質粒與pcDNA.3.1/myc-His(-)B載體質粒均以KpnI和XhoI分步雙酶切，經(jīng)連接酶作用后，，轉化至感受態(tài)E.coliTop10，氨芐青霉素篩選，挑取陽性克隆并酶切鑒定，得pcDNA.3.1/myc-His(-)B-MD2載體質粒。④將pcDNA.3.1/myc-His(-)B-TLR4-1和pcDNA.3.1/myc-His(-)B-MD2通過電穿孔轉染法轉入HEK293細胞，在mRNA水平觀察TLR4及MD2基因的表達。 結果①利用RT-PCR成功獲得人TLR4-1、TLR4-2及MD2的cDNA，構建pMD-19T-TLR4-1、pMD-19T-TLR4-2和pMD-19T-MD2質粒載體經(jīng)酶切鑒定、測序及BLAST分析，得到重組質粒。②構建pcDNA.3.1/myc-His(-)B-TLR4和pcDNA.3.1/myc-His(-)B-MD2質粒載體經(jīng)酶切鑒定、測序及BLAST分析，得到重組表達載體。③通過電穿孔轉染法，將pcDNA.3.1/myc-His(-)B-TLR4-1和pcDNA.3.1/myc-His(-)B-MD2轉染HEK293細胞后，在mRNA水平證明： TLR4和MD2基因有表達。 結論成功構建表達Toll樣受體及與部分熱原反應相關基因的表達載體，即人TLR4和MD2基因的真核表達載體pcDNA.3.1/myc-His(-)B-TLR4和pcDNA.3.1/myc-His(-)B-MD2，為構建人TLR4及MD2基因敲入細胞學模型做前期準備。
[Abstract]:Objective to construct a cytological model for expressing Toll like receptors and genes related to pyrogen reaction in vitro by genetic engineering and molecular biology, and to provide a laboratory tool for detecting the pyrogen reaction of injection.
Methods human Toll like receptor 4 (TLR4) and myeloid differentiation protein (MD2) were cloned, and pcDNA.3.1/myc-His (-) B-TLR4, pcDNA.3.1/myc-His (-) B-MD2 eukaryotic expression vector was constructed.
(1) construction and identification of pMD19-T-TLR4, pMD19-T-MD2 cloning vector: extracting the total RNA of human peripheral blood mononuclear cells, using RT-PCR to obtain TLR4-1, TLR4-2 and MD2 DNA. recovery PCR products to be cloned and sequenced on pMD-19T vector. Vector plasmids and pcDNA.3.1/myc-His (-) B expression vector plasmids were used for both EcoRI and XhoI double enzyme digestion. After the ligase action, the plasmid was converted to the receptive E.coliTop10, ampicillin was screened, the positive clones were selected and the enzyme was identified, and pcDNA.3.1/myc-His (-) B-TLR4-2 loaded particles were obtained. The qualified pcDNA.3.1/myc-His (-) B-TLR4-2 vector plasmid and P were identified. MD19-T-TLR4-1 vector plasmids were simultaneously digested with hpaI and BamHI. After the ligase action, the plasmid was transformed into the receptive E.coliTop10, ampicillin screening, the positive clones were selected and the enzyme was identified, and the pcDNA.3.1/myc-His (-) B-TLR4 vector plasmid was finally obtained. (3) the construction and identification of pcDNA.3.1/myc-His (-) B-MD2 expression vector: pMD19-T-MD2 carrying constitution The plasmid and pcDNA.3.1/myc-His (-) B vector were cut by KpnI and XhoI. After the ligase action, the plasmid was transformed to the receptive E.coliTop10, ampicillin was screened, the positive clones were selected and the pcDNA.3.1/myc-His (-) B-MD2 carrier plasmid was identified. (4) pcDNA.3.1/ myc-His (-) B-TLR4-1 and pcDNA.3.1/myc-His (-) B-MD2 through electroporation. The transfection method was transferred to HEK293 cells, and the expression of TLR4 and MD2 genes were observed at mRNA level.
Results (1) TLR4-1, TLR4-2 and MD2 cDNA were successfully obtained by RT-PCR, pMD-19T-TLR4-1, pMD-19T-TLR4-2 and pMD-19T-MD2 plasmid vectors were identified by enzyme digestion, sequencing and BLAST analysis, and recombinant plasmids were obtained. 2. Construction of pcDNA.3.1/myc-His (-) B-TLR4 and pcDNA.3.1/myc-His (-) plasmid vectors were identified by enzyme digestion, sequencing and analysis. Recombinant expression vector. (3) transfection of pcDNA.3.1/myc-His (-) B-TLR4-1 and pcDNA.3.1/myc-His (-) B-MD2 into HEK293 cells by electroporation transfection. The expression of TLR4 and MD2 genes was demonstrated at the mRNA level.
Conclusion the expression vector expressing Toll like receptor and the gene related to partial pyrogen reaction was successfully constructed, that is, the eukaryotic expression vector of human TLR4 and MD2 gene pcDNA.3.1/myc-His (-) B-TLR4 and pcDNA.3.1/myc-His (-) B-MD2, preparation for the construction of human TLR4 and MD2 gene into the cytological model.

【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346

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