Megalin基因片段（3189-4169）的克隆及其表达_药学专业毕业论文-经管之家官网！

Megalin基因片段（3189-4169）的克隆及其表达_药学专业毕业论文

【关键词】 肾炎

【摘要】 目的 大鼠Heymann肾炎（HN）是人类膜性肾病的经典动物模型，其发病机制的研究推动了人们对膜性肾病的理解。Megalin是HN主要致病抗原，也是低密度脂蛋白受体家族成员，其胞外结构形成的4簇配体结合区域均可能存在抗原决定簇。本实验拟克隆其第2簇配体结合区域，并表达融合蛋白，为进一步研究其抗原决定簇的确切位置及其功能打下基础。方法 采用RT-PCR获得大鼠肾目的片段Megalin基因3189-4169，再与原核表达质粒pTrcHis A结合构建重组质粒，然后将重组质粒转入TOP10细菌诱导表达融合蛋白，最后用Western-Blot检测表达的融合蛋白。结果 （1）RT-PCR获得的目的片段大小为1kb，测序证实为Megalin基因3189-4169;测序报告还显示有3个突变，分别位于Megalin基因的3404、3647、3702，其中前两个突变都不影响融合蛋白的氨基酸序列，第3个突变则将赖氨酸变成谷氨酸，经分析对融合蛋白表达无明显影响。（2）构建的重组质粒用BamHⅠ和EcoRⅠ双酶切后，得到预期的2个片段4.4kb和1kb，分别表示质粒pTrcHis A和Megalin基因3189-4169。（3）用IPTG诱导TOP10（含有重组质粒）融合蛋白表达后，经Western-Blot检测发现了38kD的目的蛋白。阳性对照TOP/CAT在IPTG诱导前没有出现蛋白条带，在IPTG诱导后4h出现蛋白条带；阴性对照TOP10（不含有质粒）和TOP10（含有pTrcHis A）经IPTG诱导后均无目的条带出现。另外，融合蛋白的表达量在IPTG诱导后4~6h增加明显。结论 成功构建了重组表达质粒（带有Megalin基因3189-4169的pTrcHis A）；成功诱导了融合蛋白的表达，为进一步研究Megalin基因抗原决定簇的确切位置及研究Megalin的功能提供了基础。

【关键词】 肾炎;基因;表位;融合蛋白
【Abstract】 Objective Rat Heymann nephritis（HN）is a classic model of human membranous nephropathy.Research of its pathogenesis help understand the human membranous nephropathy.Megalin is the major antigen of HN,and it is also a member of the low density lipoprotein receptor gene family；its extracellular region can bind multiple ligands,and this extracellular region form four clusters of ligand-binding domains containing pathogenic epitopes.So we cloned the second cluster of ligand binding repeats and express the recombinant proteins containing N-Terminal 6xHis tags.This study was aimed to provide the basis to precisely locate the pathogenic epitope and help understand its function.Methods We applied reverse transcription PCR to obtain objective band（Megalin gene 3189-4169）,then constructed the recombinant plasmid by inserting this fragment into pTrcHis A.After the recombinant plasmid was transfected into TOP10,the expression of 6xHis fusion protein was induced by isopropylthio-β-D-galactoside（IPTG）.Finally Western-Blot was performed to detect the fusion protein.Results RT-PCR showed the size of object band wss 1kb.After recombinant plasmid were digested with BamHⅠand EcoRⅠ,we obtained two fragments:one size was 1kb,the other was 4.4kb.Western-Blot showed the fusion protein size was 38kD，and the yield of fusion protein was obviously increased at 4~6 hours by IPTG inducing.Conclusion We succeed in constructing recombinant plasmid and obtaining the fusion protein,which may provide basis for further study.
【Key words】 nephritis;gene;epitope;fusion protein
膜性肾病是儿童及成人常见难治性肾脏疾病之一，以上皮下免疫复合物的沉积致肾小球基底膜增厚和蛋白尿为特征；其发病机制尚不清楚，现有对膜性肾病的认识很多来自于其经典动物模型Heymann肾炎（HN）的研究。因此，对Heymann肾炎的分子免疫病理机制的研究将有助于提高对膜性肾病的认识，从而改进治疗。