论文部分内容阅读
HCoV-NL63是新近发现的人冠状病毒,对其外膜糖蛋白-棘突蛋白的表达及功能的研究仍有待深入。本研究利用天坛株痘苗病毒载体,克隆构建可表达HCoV-NL63棘突蛋白四个片段(N端棘突蛋白:S1;C端棘突蛋白:S2;受体结合区大片段:RL;受体结合区小片段:RS)的重组痘苗病毒(vJSC1175-S1;vJSC1175-S2;vJSC1175-RL;vJSC1175-RS),酶切测序证实表达载体构建正确,免疫荧光分析(IFA)各重组痘苗病毒中棘突蛋白不同片段的表达与定位,Western-Blot分析表明各种重组蛋白表达正确。分析结果显示:4种重组蛋白均能有效表达,S1、RL及RS蛋白的荧光主要分布在细胞膜上,而S2蛋白的荧光则主要分布于细胞浆,各个片段的分子量大小与文献报道相同,并可进行正确的翻译修饰(糖基化)。本研究首次采用痘苗病毒天坛株载体构建制备了表达HCoV-NL63棘突蛋白不同片段的重组痘苗病毒,为进一步分析人冠状病毒HCoV-NL63棘突蛋白的结构功能及探索其抗原性和免疫原性奠定了基础。
HCoV-NL63 is a newly discovered human coronavirus. The study of the expression and function of its outer membrane glycoprotein-spinosyn is still to be further studied. In this study, four segments of HCoV-NL63 spike protein (N-terminal spinosin: S1; C-terminal spinosin: S2; receptor binding region large segment: RL; receptor The recombinant vaccinia virus (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS) with a small fragment of binding region (RS) was confirmed by restriction enzyme digestion. The expression vector was confirmed by immunofluorescence analysis (IFA) The expression and localization of different fragments of the protein, Western-Blot analysis showed that the correct expression of various recombinant proteins. The results showed that the four recombinant proteins were efficiently expressed, the fluorescence of S1, RL and RS proteins were mainly distributed in the cell membrane, while the fluorescence of S2 protein was mainly distributed in the cytoplasm. The molecular weight of each fragment was the same as reported in the literature Correct translational modification (glycosylation) is possible. In this study, the recombinant vaccinia virus expressing different segments of HCoV-NL63 spinosin protein was constructed by using vaccinia virus Tiantan strain for the first time. In order to further analyze the structural function of human coronavirus HCoV-NL63 spike protein and explore its antigenicity and immunogenicity Foundation.