本文選題：星狀病毒　切入點(diǎn)：非結(jié)構(gòu)蛋白nsP1a　出處：《遼寧醫(yī)學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 星狀病毒（human astrovirus, HAstV）是導(dǎo)致嬰幼兒腹瀉的重要病原體，HAstV感染后，腸上皮細(xì)胞凋亡可能是導(dǎo)致腹瀉的原因之一。本課題組前期研究結(jié)果表明:HAstV誘導(dǎo)腸上皮細(xì)胞凋亡是其非結(jié)構(gòu)蛋白nsP1a造成的，而nsP1a蛋白C末端（nsP1a/4）可能為主要的凋亡結(jié)構(gòu)域。本研究在前期研究成果的基礎(chǔ)上，構(gòu)建nsP1a/4真核表達(dá)載體及6種刪除突變體，通過融合蛋白表達(dá)技術(shù)，對(duì)7種重組蛋白在體外細(xì)胞中的表達(dá)，進(jìn)行初步分析；為后續(xù)研究HAstV非結(jié)構(gòu)蛋白功能及分析功能性結(jié)構(gòu)域奠定了堅(jiān)實(shí)的基礎(chǔ)，也為研究HAstV誘導(dǎo)腸上皮細(xì)胞凋亡的分子致病機(jī)制提供研究平臺(tái)。 方法 1、采用PCR技術(shù)從真核重組表達(dá)載體pEGFP-N3-nsP1a中擴(kuò)增nsP1a C末端nsP1a/4基因序列，測(cè)序鑒定正確后，采用Xho I/BamH I分別雙酶切目的片段和pEGFP-N3質(zhì)粒，，經(jīng)回收、連接、轉(zhuǎn)化等方法構(gòu)建野生型nsP1a/4真核表達(dá)重組質(zhì)粒pEGFP-N3-nsp1a/4。 2、根據(jù)星狀病毒nsP1a/4蛋白的剪切位點(diǎn)、磷酸化位點(diǎn)及蛋白死亡結(jié)構(gòu)域等信息，設(shè)計(jì)nsP1a/4不同位點(diǎn)的截短的各種突變體的擴(kuò)增引物；不同的刪除突變?yōu)椋害?-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304，經(jīng)PCR、雙酶切、連接、轉(zhuǎn)化等方法構(gòu)建6種不同的刪除突變。 3、制備7種轉(zhuǎn)染級(jí)別的重組質(zhì)粒：pEGFP-N3-nsp1a/4、pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、 pEGFP-N3Δ1-209、 pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304。分別運(yùn)用脂質(zhì)體Lipofectamine2000轉(zhuǎn)染試劑轉(zhuǎn)染BHK21細(xì)胞，建立瞬時(shí)轉(zhuǎn)染體系，48-72小時(shí)后進(jìn)行PCR熒光觀察及Western blot檢測(cè)，對(duì)7種重組體進(jìn)行蛋白的表達(dá)鑒定。 結(jié)果 1、nsp1a/4、nsp1a/4蛋白6種突變體（Δ1-88、Δ1-176、Δ1-209、Δ53-174、Δ225-304、Δ273-304）的目的基因產(chǎn)物成功擴(kuò)增。 2、采用基因重組技術(shù)，成功構(gòu)建nsp1a/4蛋白，6種nsp1a/4刪除突變體的真核表達(dá)載體基因序列與已知基因序列完全一致，酶切結(jié)果與測(cè)序結(jié)果表明已成功插入真核表達(dá)載體pEGFP-N3中。 3、將7種重組蛋白轉(zhuǎn)染BHK21細(xì)胞，24小時(shí)后顯微鏡下觀察到EGFP綠色熒光，48小時(shí)后PCR檢測(cè)及Western blot檢測(cè)表明重組蛋白能夠高效在體外細(xì)胞中表達(dá)。 結(jié)論 成功構(gòu)建HAstV非結(jié)構(gòu)蛋白C末端nsP1a/4基因真核表達(dá)載體pEGFP-N3-nsP1a/4及nsP1a/4蛋白6種刪除突變體(pEGFP-N3Δ1-88、pEGFP-N3Δ1-176、pEGFP-N3Δ1-209、pEGFP-N3Δ53-174、pEGFP-N3Δ225-304、pEGFP-N3Δ273-304)7種重組蛋白能夠在體外細(xì)胞BHK21中高效表達(dá)，成功建立非結(jié)構(gòu)蛋白不同突變體的細(xì)胞模型，為進(jìn)一步研究nsP1a/4蛋白基因功能提供實(shí)驗(yàn)平臺(tái)。
[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.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R373

【共引文獻(xiàn)】

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1 朱攀;昆明市小兒腹瀉相關(guān)病毒的分子流行病學(xué)研究及病毒基因組克隆[D];昆明理工大學(xué);2013年

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