發(fā)熱伴血小板減少綜合征布尼亞病毒小基因組的構(gòu)建
發(fā)布時間：2019-03-07 20:32
【摘要】：背景：發(fā)熱伴血小板減少綜合征(severe fever with thrombocytopenia syndrome, SFTS)是一種以嚴重發(fā)熱伴血小板減少為主要特征的新型傳染性疾病。其病原體為發(fā)熱伴血小板減少綜合征病毒(severe fever with thrombocytopenia syndrome bunyavirus, SFTSV),這是一種新型的蜱傳蟲媒病毒。該病毒在我國于2009年在湖北、河南等地首次發(fā)現(xiàn)。SFTSV能夠引起發(fā)熱、胃腸道癥狀、血小板減少和白細胞減少的癥狀,以及多器官衰竭導致死亡。這種新發(fā)傳染性疾病平均致死率為12%,局部地區(qū)致死率高達30%.該病毒自2009年我國中部地區(qū)首次發(fā)現(xiàn)以來,在2010年又在我國中部和東北部地區(qū)再次頻繁出現(xiàn)。由于缺少有效的抗發(fā)熱伴血小板減少綜合征布尼亞病毒的藥物和疫苗,因此該病毒仍然對人類的健康存在嚴重的威脅。 目的：本實驗通過構(gòu)建SFTSV小基因組,來研究病毒基因的功能,轉(zhuǎn)錄及復制過程。由于SFTSV轉(zhuǎn)錄復制所需原件多,本實驗還通過構(gòu)建穩(wěn)定細胞系的方法來提高轉(zhuǎn)染效率,增加小基因組成功的可能性。由于需要檢測質(zhì)粒轉(zhuǎn)染哺乳動物細胞后的表達情況,我們對SFTSV基因組L、M片段進行了一系列的截短表達,尋找出穩(wěn)定的蛋白制備抗體,以便對SFTSV各片段的表達進行檢測。本實驗將反向遺傳技術(shù)的手段用于該病毒的研究,將為今后藥物篩選,疫苗制備的工作打好基礎。 方法： 1.小基因組的構(gòu)建：構(gòu)建分別帶有SFTSV S, M, L片段5’和3’端UTR區(qū)并在中間插入報告基因egfp的重組質(zhì)粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S;將重組質(zhì)粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S分別與表達NP和RdRp蛋白的重組質(zhì)粒pcDNA-NP、 pcDNA-L組合轉(zhuǎn)染哺乳動物細胞,通過報告基因egfp的表達水平的差異對小基因組成功與否及效率進行評價。 2.穩(wěn)定表達SFTSV NP蛋白細胞系的篩選：通過G418對293細胞的毒性評估實驗,選擇合適的G418濃度用于后續(xù)細胞系的篩選；將表達SFTSV NP并帶有6418抗性基因的重組質(zhì)粒轉(zhuǎn)染293細胞,以稀釋法篩選出穩(wěn)定表達該蛋白的細胞系；對該細胞系進行鑒定； 3.原核表達SFTSV RdRp、Gn、Gc蛋白：設計相應引物,構(gòu)建各個蛋白的原核表達重組質(zhì)粒；將陽性重組質(zhì)粒轉(zhuǎn)化大腸桿菌,小量誘導表達蛋白。 結(jié)果： 1.獲得插入報告基因egfp的各重組質(zhì)粒pcDNA-TP-L、pcDNA-TP-M、pcDNA-TP-S; 2.將帶有報告基因egfp的各重組質(zhì)粒與表達表達NP和RdRp蛋白的重組質(zhì)粒pcDNA-NP、pcDNA-L組合轉(zhuǎn)染哺乳動物細胞,報告基因egfp表達出綠色熒光； 3.獲得穩(wěn)定表達SFTSV NP蛋白的293細胞系； 4.獲得SFTSV結(jié)構(gòu)蛋白的原核表達。 結(jié)論：發(fā)熱伴血小板減少綜合征布尼亞病毒的小基因組構(gòu)建初步建立,并且在穩(wěn)定表達SFTSV NP的細胞系中轉(zhuǎn)染能提高轉(zhuǎn)染效率,SFTSV的結(jié)構(gòu)蛋白可在大腸桿菌中原核表達。
[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.
【學位授予單位】：湖北大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：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.
【學位授予單位】：湖北大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R510
【相似文獻】
相關(guān)期刊論文 前10條
1 龍志國;朱紅波;章正華;姜鏵;夏云金;萬楚成;張霞;;新型布尼亞病毒感染11例臨床分析[J];內(nèi)科急危重癥雜志;2011年06期
2 徐學靜;夏永泉;張葵;;新型布尼亞病毒感染致發(fā)熱伴血小板減少1例[J];臨床檢驗雜志;2012年05期
3 徐慧寧;張宇;于杰;高巍;;老年新型布尼亞病毒致發(fā)熱伴血小板減少綜合征5例[J];中國老年學雜志;2012年18期
4 杜景華;徐慧寧;劉s，
本文編號：2436437
本文鏈接：http://sikaile.net/yixuelunwen/chuanranbingxuelunwen/2436437.html
最近更新
教材專著
