星状病毒非结构蛋白nspla C末端7种突变体的构建与表达分析

发布时间：2018-03-14 22:18

本文选题：星状病毒　切入点：非结构蛋白nsP1a　出处：《辽宁医学院》2013年硕士论文　论文类型：学位论文

【摘要】：目的星状病毒（human astrovirus, HAstV）是导致婴幼儿腹泻的重要病原体，HAstV感染后，肠上皮细胞凋亡可能是导致腹泻的原因之一。本课题组前期研究结果表明:HAstV诱导肠上皮细胞凋亡是其非结构蛋白nsP1a造成的，而nsP1a蛋白C末端（nsP1a/4）可能为主要的凋亡结构域。本研究在前期研究成果的基础上，构建nsP1a/4真核表达载体及6种删除突变体，通过融合蛋白表达技术，对7种重组蛋白在体外细胞中的表达，进行初步分析；为后续研究HAstV非结构蛋白功能及分析功能性结构域奠定了坚实的基础，也为研究HAstV诱导肠上皮细胞凋亡的分子致病机制提供研究平台。方法 1、采用PCR技术从真核重组表达载体pEGFP-N3-nsP1a中扩增nsP1a C末端nsP1a/4基因序列，测序鉴定正确后，采用Xho I/BamH I分别双酶切目的片段和pEGFP-N3质粒，，经回收、连接、转化等方法构建野生型nsP1a/4真核表达重组质粒pEGFP-N3-nsp1a/4。 2、根据星状病毒nsP1a/4蛋白的剪切位点、磷酸化位点及蛋白死亡结构域等信息，设计nsP1a/4不同位点的截短的各种突变体的扩增引物；不同的删除突变为：Δ1-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304，经PCR、双酶切、连接、转化等方法构建6种不同的删除突变。 3、制备7种转染级别的重组质粒：pEGFP-N3-nsp1a/4、pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、 pEGFP-N3Δ1-209、 pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304。分别运用脂质体Lipofectamine2000转染试剂转染BHK21细胞，建立瞬时转染体系，48-72小时后进行PCR荧光观察及Western blot检测，对7种重组体进行蛋白的表达鉴定。结果 1、nsp1a/4、nsp1a/4蛋白6种突变体（Δ1-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304）的目的基因产物成功扩增。 2、采用基因重组技术，成功构建nsp1a/4蛋白，6种nsp1a/4删除突变体的真核表达载体基因序列与已知基因序列完全一致，酶切结果与测序结果表明已成功插入真核表达载体pEGFP-N3中。 3、将7种重组蛋白转染BHK21细胞，24小时后显微镜下观察到EGFP绿色荧光，48小时后PCR检测及Western blot检测表明重组蛋白能够高效在体外细胞中表达。结论成功构建HAstV非结构蛋白C末端nsP1a/4基因真核表达载体pEGFP-N3-nsP1a/4及nsP1a/4蛋白6种删除突变体(pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、pEGFP-N3Δ1-209、pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304)7种重组蛋白能够在体外细胞BHK21中高效表达，成功建立非结构蛋白不同突变体的细胞模型，为进一步研究nsP1a/4蛋白基因功能提供实验平台。
[Abstract]:Purpose. Stellate virus human astrovirus (HAstV) is an important pathogen leading to infantile diarrhea after infection with HAstV. Apoptosis of intestinal epithelial cells may be one of the causes of diarrhea. Our previous research results show that the apoptosis of intestinal epithelial cells induced by 1: HASTV is caused by its non-structural protein nsP1a. In this study, nsP1a/4 eukaryotic expression vector and 6 deleted mutants were constructed on the basis of previous research results, and expressed by fusion protein. The expression of seven recombinant proteins in vitro was preliminarily analyzed, which laid a solid foundation for the further study of the function of HAstV non-structural proteins and the analysis of functional domains. It also provides a platform for studying the molecular pathogenetic mechanism of HAstV induced apoptosis of intestinal epithelial cells. Method. 1. The nsP1a C-terminal nsP1a/4 gene sequence was amplified by PCR from eukaryotic recombinant expression vector pEGFP-N3-nsP1a. After sequencing, the target fragment and pEGFP-N3 plasmid were digested with Xho I / BamH I, respectively, and were recovered and ligated. The wild type nsP1a/4 eukaryotic expression plasmid pEGFP-N3-nsp1a / 4 was constructed by transformation. 2According to the information of splicing site, phosphorylation site and protein death domain of stellate virus nsP1a/4 protein, primers for amplification of truncated mutants of different nsP1a/4 sites were designed, and the different deletion mutants were: 螖 1-88, 螖 1-176, 螖 1-209, 螖 53-174, 螖 225-304, 螖 273-304. Six different deletion mutations were constructed by transformation. 3. Seven kinds of recombinant plasmids: pEGFP-N3-nsp1a- / 4pEGFP-N3 螖 1-88pEGFP-N3 螖 1-176, pEGFP-N3 螖 1-209, pEGFP-N3 螖 53-174pEGFP-N3 螖 225-304pEGFP-N3 螖 273-304 were prepared. The BHK21 cells were transfected with liposome Lipofectamine2000 transfection reagent. After 48 to 72 hours of transient transfection, PCR fluorescence observation and Western blot detection were performed. Results. The target gene products of 6 mutants (螖 1-88, 螖 1-176, 螖 1-209, 螖 53-174, 螖 225-304, 螖 273-304) were amplified successfully. 2. The eukaryotic expression vector sequence of 6 nsp1a/4 deleted mutants of nsp1a/4 protein was successfully constructed by gene recombination technique. The results of restriction endonuclease digestion and sequencing showed that the eukaryotic expression vector pEGFP-N3 had been successfully inserted into the eukaryotic expression vector. 3After the 7 recombinant proteins were transfected into BHK21 cells for 24 hours, PCR detection and Western blot detection showed that the recombinant proteins could be expressed efficiently in vitro. Conclusion. The eukaryotic expression vector pEGFP-N3 螖 1-88pEGFP-N3 螖 1-176pEGFP-N3 螖 1-206 pEGFP-N3 螖 53-174pEGFP-N3 螖 225-304pEGFP-N3 螖 273-304pEGFP-N3 螖 273-304were successfully constructed, and the cell models of different mutants of non-structural proteins were successfully established. It provides an experimental platform for further study on the function of nsP1a/4 protein gene.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R373

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