发热伴血小板减少综合征布尼亚病毒小基因组的构建
发布时间：2019-03-07 20:32
【摘要】：背景：发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome, SFTS)是一种以严重发热伴血小板减少为主要特征的新型传染性疾病。其病原体为发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome bunyavirus, SFTSV),这是一种新型的蜱传虫媒病毒。该病毒在我国于2009年在湖北、河南等地首次发现。SFTSV能够引起发热、胃肠道症状、血小板减少和白细胞减少的症状,以及多器官衰竭导致死亡。这种新发传染性疾病平均致死率为12%,局部地区致死率高达30%.该病毒自2009年我国中部地区首次发现以来,在2010年又在我国中部和东北部地区再次频繁出现。由于缺少有效的抗发热伴血小板减少综合征布尼亚病毒的药物和疫苗,因此该病毒仍然对人类的健康存在严重的威胁。 目的：本实验通过构建SFTSV小基因组,来研究病毒基因的功能,转录及复制过程。由于SFTSV转录复制所需原件多,本实验还通过构建稳定细胞系的方法来提高转染效率,增加小基因组成功的可能性。由于需要检测质粒转染哺乳动物细胞后的表达情况,我们对SFTSV基因组L、M片段进行了一系列的截短表达,寻找出稳定的蛋白制备抗体,以便对SFTSV各片段的表达进行检测。本实验将反向遗传技术的手段用于该病毒的研究,将为今后药物筛选,疫苗制备的工作打好基础。 方法： 1.小基因组的构建：构建分别带有SFTSV S, M, L片段5’和3’端UTR区并在中间插入报告基因egfp的重组质粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S;将重组质粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S分别与表达NP和RdRp蛋白的重组质粒pcDNA-NP、 pcDNA-L组合转染哺乳动物细胞,通过报告基因egfp的表达水平的差异对小基因组成功与否及效率进行评价。 2.稳定表达SFTSV NP蛋白细胞系的筛选：通过G418对293细胞的毒性评估实验,选择合适的G418浓度用于后续细胞系的筛选；将表达SFTSV NP并带有6418抗性基因的重组质粒转染293细胞,以稀释法筛选出稳定表达该蛋白的细胞系；对该细胞系进行鉴定； 3.原核表达SFTSV RdRp、Gn、Gc蛋白：设计相应引物,构建各个蛋白的原核表达重组质粒；将阳性重组质粒转化大肠杆菌,小量诱导表达蛋白。 结果： 1.获得插入报告基因egfp的各重组质粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S; 2.将带有报告基因egfp的各重组质粒与表达表达NP和RdRp蛋白的重组质粒pcDNA-NP、pcDNA-L组合转染哺乳动物细胞,报告基因egfp表达出绿色荧光； 3.获得稳定表达SFTSV NP蛋白的293细胞系； 4.获得SFTSV结构蛋白的原核表达。 结论：发热伴血小板减少综合征布尼亚病毒的小基因组构建初步建立,并且在稳定表达SFTSV NP的细胞系中转染能提高转染效率,SFTSV的结构蛋白可在大肠杆菌中原核表达。
[Abstract]:Background: fever with thrombocytopenia syndrome (severe fever with thrombocytopenia syndrome, SFTS) is a new infectious disease characterized by severe fever with thrombocytopenia. The pathogen is fever with thrombocytopenia syndrome virus (severe fever with thrombocytopenia syndrome bunyavirus, SFTSV), a new tick-borne insect-borne virus. SFTSV can cause fever, gastrointestinal symptoms, thrombocytopenia and leukopenia, as well as multiple organ failure leading to death. The average fatality rate of the new infectious disease is 12%, while local mortality is as high as 30%. Since the virus was first discovered in the central region of China in 2009, it has reappeared frequently in the central and northeast regions of China in 2010. Due to the lack of effective drugs and vaccines against Bunia virus with fever and thrombocytopenia syndrome, the virus remains a serious threat to human health. Aim: to study the function, transcription and replication of viral genes by constructing a small genome of SFTSV. In order to improve the transfection efficiency and increase the possibility of success of small genome, the method of constructing stable cell lines was used to improve the transfection efficiency and increase the possibility of success of small genome because of the large number of SFTSV transcription and replication. Because of the need to detect the expression of plasmid transfected mammalian cells, we have carried out a series of truncated expression of L and M fragment of SFTSV genome, and found out the stable protein to prepare antibody to detect the expression of each fragment of SFTSV. In this experiment, reverse genetic technique is applied to the study of the virus, which will lay a good foundation for drug screening and vaccine preparation in the future. Methods: 1. Construction of small genome: construction of recombinant plasmid pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S; with 5 'and 3'-terminal UTR regions of SFTSV S, M, L fragment and insertion of reporter gene egfp in the middle Mammalian cells were transfected with recombinant plasmid pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S and recombinant plasmid pcDNA-NP, pcDNA-L expressing NP and RdRp protein, respectively. The success and efficiency of small genome were evaluated by the difference of the expression level of reporter gene egfp. 2. Screening of cell lines stably expressing SFTSV NP protein: by evaluating the toxicity of G418 to 293 cells, the suitable concentration of G418 was selected for the selection of subsequent cell lines. The recombinant plasmid expressing SFTSV NP and carrying 6418 resistance gene was transfected into 293 cells, and the stable expression cell line was screened by dilution method, the cell line was identified, and the cell line was identified. Prokaryotic expression of SFTSV RdRp,Gn,Gc protein: the corresponding primers were designed to construct the prokaryotic expression recombinant plasmid of each protein, and the positive recombinant plasmid was transformed into E. coli to induce the expression of the protein in a small amount. Results: 1. The recombinant plasmids pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S; 2. Inserted into the reporter gene egfp were obtained. The recombinant plasmids with reporter gene egfp and recombinant plasmid pcDNA-NP,pcDNA-L expressing NP and RdRp proteins were combined to transfect mammalian cells, and the reporter gene egfp expressed green fluorescence. The stable expression of SFTSV NP protein was obtained in 293 cell lines; 4. The prokaryotic expression of SFTSV structural protein was obtained. Conclusion: the construction of a small genome of Bunia virus with fever and thrombocytopenia syndrome was established and transfected into a stable cell line expressing SFTSV NP could improve the transfection efficiency. The structural protein of SFTSV could be expressed in E. coli.
【学位授予单位】：湖北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R510
[Abstract]:Background: fever with thrombocytopenia syndrome (severe fever with thrombocytopenia syndrome, SFTS) is a new infectious disease characterized by severe fever with thrombocytopenia. The pathogen is fever with thrombocytopenia syndrome virus (severe fever with thrombocytopenia syndrome bunyavirus, SFTSV), a new tick-borne insect-borne virus. SFTSV can cause fever, gastrointestinal symptoms, thrombocytopenia and leukopenia, as well as multiple organ failure leading to death. The average fatality rate of the new infectious disease is 12%, while local mortality is as high as 30%. Since the virus was first discovered in the central region of China in 2009, it has reappeared frequently in the central and northeast regions of China in 2010. Due to the lack of effective drugs and vaccines against Bunia virus with fever and thrombocytopenia syndrome, the virus remains a serious threat to human health. Aim: to study the function, transcription and replication of viral genes by constructing a small genome of SFTSV. In order to improve the transfection efficiency and increase the possibility of success of small genome, the method of constructing stable cell lines was used to improve the transfection efficiency and increase the possibility of success of small genome because of the large number of SFTSV transcription and replication. Because of the need to detect the expression of plasmid transfected mammalian cells, we have carried out a series of truncated expression of L and M fragment of SFTSV genome, and found out the stable protein to prepare antibody to detect the expression of each fragment of SFTSV. In this experiment, reverse genetic technique is applied to the study of the virus, which will lay a good foundation for drug screening and vaccine preparation in the future. Methods: 1. Construction of small genome: construction of recombinant plasmid pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S; with 5 'and 3'-terminal UTR regions of SFTSV S, M, L fragment and insertion of reporter gene egfp in the middle Mammalian cells were transfected with recombinant plasmid pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S and recombinant plasmid pcDNA-NP, pcDNA-L expressing NP and RdRp protein, respectively. The success and efficiency of small genome were evaluated by the difference of the expression level of reporter gene egfp. 2. Screening of cell lines stably expressing SFTSV NP protein: by evaluating the toxicity of G418 to 293 cells, the suitable concentration of G418 was selected for the selection of subsequent cell lines. The recombinant plasmid expressing SFTSV NP and carrying 6418 resistance gene was transfected into 293 cells, and the stable expression cell line was screened by dilution method, the cell line was identified, and the cell line was identified. Prokaryotic expression of SFTSV RdRp,Gn,Gc protein: the corresponding primers were designed to construct the prokaryotic expression recombinant plasmid of each protein, and the positive recombinant plasmid was transformed into E. coli to induce the expression of the protein in a small amount. Results: 1. The recombinant plasmids pcDNA-TP-L,pcDNA-TP-M,pcDNA-TP-S; 2. Inserted into the reporter gene egfp were obtained. The recombinant plasmids with reporter gene egfp and recombinant plasmid pcDNA-NP,pcDNA-L expressing NP and RdRp proteins were combined to transfect mammalian cells, and the reporter gene egfp expressed green fluorescence. The stable expression of SFTSV NP protein was obtained in 293 cell lines; 4. The prokaryotic expression of SFTSV structural protein was obtained. Conclusion: the construction of a small genome of Bunia virus with fever and thrombocytopenia syndrome was established and transfected into a stable cell line expressing SFTSV NP could improve the transfection efficiency. The structural protein of SFTSV could be expressed in E. coli.
【学位授予单位】：湖北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R510
【相似文献】
相关期刊论文 前10条
1 龙志国;朱红波;章正华;姜铧;夏云金;万楚成;张霞;;新型布尼亚病毒感染11例临床分析[J];内科急危重症杂志;2011年06期
2 徐学静;夏永泉;张葵;;新型布尼亚病毒感染致发热伴血小板减少1例[J];临床检验杂志;2012年05期
3 徐慧宁;张宇;于杰;高巍;;老年新型布尼亚病毒致发热伴血小板减少综合征5例[J];中国老年学杂志;2012年18期
4 杜景华;徐慧宁;刘s，
本文编号：2436437
本文链接：https://www.wllwen.com/yixuelunwen/chuanranbingxuelunwen/2436437.html
最近更新
教材专著
