人TLR4及MD2基因真核表达载体的构建

发布时间：2018-05-05 13:56

本文选题：TLR4 + MD2　；参考：《宁夏医科大学》2012年硕士论文

【摘要】：目的采用基因工程和分子生物学的方法，构建体外表达Toll样受体及与热原反应相关基因的细胞学模型，为检测注射剂的热原反应提供实验室使用工具。方法克隆人Toll样受体4（TLR4）和髓样分化蛋白（MD2），构建pcDNA.3.1/myc-His(-)B-TLR4、pcDNA.3.1/myc-His(-)B-MD2真核表达载体。 ①pMD19-T-TLR4、pMD19-T-MD2克隆载体的构建及鉴定：提取人外周血单核细胞总RNA，利用RT-PCR得到TLR4-1、TLR4-2和MD2的DNA。回收PCR产物连接于pMD-19T载体上进行克隆和测序。②pcDNA.3.1/myc-His(-)B-TLR4表达载体的构建及鉴定：测序鉴定后的pMD19-T-TLR4-2载体质粒与pcDNA.3.1/myc-His(-)B表达载体质粒同时进行EcoRI和XhoI双酶切，经连接酶作用后，转化至感受态E.coliTop10，氨苄青霉素筛选，挑取阳性克隆并酶切鉴定，得pcDNA.3.1/myc-His(-)B-TLR4-2载体质粒。鉴定合格的pcDNA.3.1/myc-His(-)B-TLR4-2载体质粒与pMD19-T-TLR4-1载体质粒同时进行hpaI和BamHI双酶切，经连接酶作用后，转化至感受态E.coliTop10，氨苄青霉素筛选，挑取阳性克隆并酶切鉴定，最终获得pcDNA.3.1/myc-His(-)B-TLR4载体质粒。③pcDNA.3.1/myc-His(-)B-MD2表达载体的构建及鉴定：pMD19-T-MD2载体质粒与pcDNA.3.1/myc-His(-)B载体质粒均以KpnI和XhoI分步双酶切，经连接酶作用后，，转化至感受态E.coliTop10，氨苄青霉素筛选，挑取阳性克隆并酶切鉴定，得pcDNA.3.1/myc-His(-)B-MD2载体质粒。④将pcDNA.3.1/myc-His(-)B-TLR4-1和pcDNA.3.1/myc-His(-)B-MD2通过电穿孔转染法转入HEK293细胞，在mRNA水平观察TLR4及MD2基因的表达。结果①利用RT-PCR成功获得人TLR4-1、TLR4-2及MD2的cDNA，构建pMD-19T-TLR4-1、pMD-19T-TLR4-2和pMD-19T-MD2质粒载体经酶切鉴定、测序及BLAST分析，得到重组质粒。②构建pcDNA.3.1/myc-His(-)B-TLR4和pcDNA.3.1/myc-His(-)B-MD2质粒载体经酶切鉴定、测序及BLAST分析，得到重组表达载体。③通过电穿孔转染法，将pcDNA.3.1/myc-His(-)B-TLR4-1和pcDNA.3.1/myc-His(-)B-MD2转染HEK293细胞后，在mRNA水平证明： TLR4和MD2基因有表达。结论成功构建表达Toll样受体及与部分热原反应相关基因的表达载体，即人TLR4和MD2基因的真核表达载体pcDNA.3.1/myc-His(-)B-TLR4和pcDNA.3.1/myc-His(-)B-MD2，为构建人TLR4及MD2基因敲入细胞学模型做前期准备。
[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.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

【参考文献】