SFTSV全基因组序列及其核衣壳蛋白的原核表达分析

发布时间：2018-06-01 13:32

本文选题：SFTSV + 全基因组测序　；参考：《浙江师范大学》2014年硕士论文

【摘要】：发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV)自2011年被报道以来,在我国流行范围有扩大趋势。该病毒感染引起的发热伴血小板减少综合症(Severe fever with thrombocytopenia syndrome, SFTS)主要表现为发热、血小板减少,严重者出现多器官功能衰竭进而死亡。由于SFTS易感性高,对于SFTSV的研究也处于初级阶段,尚无有效的预防及治疗的措施,研究SFTSV对于有效预防与控制SFTS的暴发流行具有重要理论与实践指导意义。本研究主要目的为建立SFTSV的荧光定量RT-PCR检测方法,从浙江省SFTSV感染阳性血清标本中分离SFTSV,并对分离株进行全基因组测序和分子进化分析及分子流行病学分析,初步了解浙江地区SFTSV的区域进化特征。由于布尼亚科病毒感染宿主能快速诱导核衣壳(N)蛋白抗体的形成,因此,本研究克隆SFTSV的N蛋白基因,对重组表达质粒进行原核表达,并对重组蛋白进行纯化和生化鉴定。期望获得具有免疫学活性的纯化重组N蛋白,为临床诊断、多抗或单抗制备提供重要抗原,也为SFTSV血清学检测和工程疫苗研制奠定前期基础。主要研究结果如下：针对新型布尼亚病毒基因RdRP的保守区设计特异性引物和TaqMan探针,建立了检测SFTSV的TagMan实时荧光定量RT-PCR检测方法。建立了SFTSV在非洲绿猴肾细胞中的培养与分离鉴定方法。对分离到SFTSV浙江毒株Zhejiang/01/2011进行鉴定,并提取RNA基因组；用RT-PCR技术扩增覆盖全基因组的17个重叠基因片段,测序并拼接出全基因组序列,结果表明,SFTSV浙江毒株全基因组cDNA序列共分3段,S片段为1745bp,M片段为3378bp,L片段为6368bp。由于这些引物位于病毒基因组相对保守的区域,所以可以用于其他未知序列SFTSV的全基因组测序。该病毒株序列已上传GenBank,S片段登录号为KJ597823,M片段登陆号为KJ597824,L片段登录号为KJ597825。从GenBank数据库中获取各地SFTSV全基因组序列,采用RAxML软件及MEGA软件对分离株基因组核苷酸序列与GeneBank已经公布的SFTSV核苷酸序列和白蛉病毒属部分其他病毒进行比对,并构建进化树,进行同源性分析。序列分析结果表明,SFTSV浙江分离株Zhejiang/01/2011与已报道的日本分离株在同一分支,相似性较大,其中与Japan/SPL004A/2013毒株相似性最高,S片段相似性为98%,M片段为97%,L片段为98%；与国内其他地区分离株不在同一分支,差异性较大。同时,比对结果也证实该毒株属于白蛉病毒属。运用RT-PCR和分子克隆技术,成功分离位于SFTSV S片段的N蛋白全长基因(738bp),构建了克隆载体pMD-19T-SFTSV-NP和原核表达载体pET-42a(+)-SFTSV-NP,并经测序分析。将原核表达载体pET-42a(+)-SFTSV-NP转化宿主菌BL21(DE3),优化了IPTG诱导表达体系,目的重组蛋白获得高效表达。SDS-PAGE电泳分析表明,目的蛋白以包涵体表达为主。对重组蛋白进行镍柱亲和层析,Western blot分析结果表明,重组蛋白具有良好的抗原性,为SFTSV的早期诊断奠定了生化基础。
[Abstract]:Fever accompanied by thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV) has been expanded in our country since 2011, and the fever associated with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome) is the main manifestation of the virus. Fever, thrombocytopenia, multiple organ failure and death in serious cases. Because of the high susceptibility of SFTS, the research on SFTSV is also in the primary stage, and there is no effective prevention and treatment measures. The study of SFTSV has important theoretical and practical significance for the effective prevention and control of the outbreak of SFTS.
The main purpose of this study was to establish a SFTSV fluorescence quantitative RT-PCR detection method, to isolate SFTSV from the positive serum samples of SFTSV infection in Zhejiang Province, and to carry out complete genome sequencing, molecular evolution analysis and molecular epidemiological analysis on the isolates, and to preliminarily understand the regional evolution characteristics of SFTSV in the Zhejiang region. The host can quickly induce the formation of Nucleocapsid (N) protein antibodies. Therefore, this study cloned the N protein gene of SFTSV, expressed the recombinant expression plasmid, purified the recombinant protein and identified the recombinant protein, and hoped to obtain the purified recombinant N protein with immunological activity to provide important antigen for the clinical diagnosis and the preparation of polyclonal or monoclonal antibodies. The research work has laid a foundation for SFTSV serological detection and engineering vaccine development.
Based on the design of specific primers and TaqMan probes in the conservative region of the new type of RdRP virus gene, a real-time TagMan fluorescence quantitative RT-PCR detection method for detecting SFTSV was established. The culture and isolation identification method of SFTSV in the renal cell of African green monkey was established. The isolated SFTSV Zhejiang strain Zhejiang/01/2011 was identified and the RNA base was extracted. RT-PCR technique was used to amplify 17 overlapping gene fragments covering the whole genome and sequenced and spliced the whole genome sequence. The results showed that the whole genome cDNA sequence of SFTSV Zhejiang strains was divided into 3 segments, S fragments were 1745bp, M fragments were 3378bp, L fragments were 6368bp. because they were in the relatively conservative region of the virus genome, so they could be located in the relatively conservative region of the virus genome. Whole genome sequencing for other unknown sequence SFTSV. The virus sequence has been uploaded GenBank, the S fragment login number is KJ597823, the M fragment landing number is KJ597824, the L fragment logon number is KJ597825. from the GenBank database to obtain the whole genome sequence of SFTSV, and uses RAxML software and MEGA software for the nucleotide sequence of the isolates. NeBank the SFTSV nucleotide sequence has been compared with some other viruses of the genus, and constructed the evolutionary tree for homology analysis. The sequence analysis showed that the SFTSV Zhejiang isolate Zhejiang/01/2011 was similar to the reported Japanese isolated strain in the same branch, which was most similar to the Japan/SPL004A/2013 strain. The S fragment similarity was 98%, the M fragment was 97%, the L fragment was 98%, and the isolated strains were not in the same branch in other regions of China.
The full-length N protein gene (738bp) in the SFTSV S fragment was successfully separated by RT-PCR and molecular cloning technology, and the clone carrier pMD-19T-SFTSV-NP and the prokaryotic expression vector pET-42a (+) -SFTSV-NP were constructed. The primary expression vector pET-42a (+) -SFTSV-NP was converted to BL21 (DE3), and the aim was to optimize the induction expression system. The expression of histone by high expression.SDS-PAGE electrophoresis showed that the target protein was mainly expressed as inclusion body. The recombinant protein was analyzed by nickel column affinity chromatography. The results of Western blot analysis showed that the recombinant protein had good antigenicity and laid a biochemical basis for the early diagnosis of SFTSV.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R373.3

【参考文献】