本文選題：SFTSV + 全基因組測序��； 參考：《浙江師范大學(xué)》2014年碩士論文

【摘要】：發(fā)熱伴血小板減少綜合征布尼亞病毒(Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV)自2011年被報道以來,在我國流行范圍有擴(kuò)大趨勢。該病毒感染引起的發(fā)熱伴血小板減少綜合癥(Severe fever with thrombocytopenia syndrome, SFTS)主要表現(xiàn)為發(fā)熱、血小板減少,嚴(yán)重者出現(xiàn)多器官功能衰竭進(jìn)而死亡。由于SFTS易感性高,對于SFTSV的研究也處于初級階段,尚無有效的預(yù)防及治療的措施,研究SFTSV對于有效預(yù)防與控制SFTS的暴發(fā)流行具有重要理論與實踐指導(dǎo)意義。 本研究主要目的為建立SFTSV的熒光定量RT-PCR檢測方法,從浙江省SFTSV感染陽性血清標(biāo)本中分離SFTSV,并對分離株進(jìn)行全基因組測序和分子進(jìn)化分析及分子流行病學(xué)分析,初步了解浙江地區(qū)SFTSV的區(qū)域進(jìn)化特征。由于布尼亞科病毒感染宿主能快速誘導(dǎo)核衣殼(N)蛋白抗體的形成,因此,本研究克隆SFTSV的N蛋白基因,對重組表達(dá)質(zhì)粒進(jìn)行原核表達(dá),并對重組蛋白進(jìn)行純化和生化鑒定。期望獲得具有免疫學(xué)活性的純化重組N蛋白,為臨床診斷、多抗或單抗制備提供重要抗原,也為SFTSV血清學(xué)檢測和工程疫苗研制奠定前期基礎(chǔ)。主要研究結(jié)果如下： 針對新型布尼亞病毒基因RdRP的保守區(qū)設(shè)計特異性引物和TaqMan探針,建立了檢測SFTSV的TagMan實時熒光定量RT-PCR檢測方法。建立了SFTSV在非洲綠猴腎細(xì)胞中的培養(yǎng)與分離鑒定方法。對分離到SFTSV浙江毒株Zhejiang/01/2011進(jìn)行鑒定,并提取RNA基因組；用RT-PCR技術(shù)擴(kuò)增覆蓋全基因組的17個重疊基因片段,測序并拼接出全基因組序列,結(jié)果表明,SFTSV浙江毒株全基因組cDNA序列共分3段,S片段為1745bp,M片段為3378bp,L片段為6368bp。由于這些引物位于病毒基因組相對保守的區(qū)域,所以可以用于其他未知序列SFTSV的全基因組測序。該病毒株序列已上傳GenBank,S片段登錄號為KJ597823,M片段登陸號為KJ597824,L片段登錄號為KJ597825。從GenBank數(shù)據(jù)庫中獲取各地SFTSV全基因組序列,采用RAxML軟件及MEGA軟件對分離株基因組核苷酸序列與GeneBank已經(jīng)公布的SFTSV核苷酸序列和白蛉病毒屬部分其他病毒進(jìn)行比對,并構(gòu)建進(jìn)化樹,進(jìn)行同源性分析。序列分析結(jié)果表明,SFTSV浙江分離株Zhejiang/01/2011與已報道的日本分離株在同一分支,相似性較大,其中與Japan/SPL004A/2013毒株相似性最高,S片段相似性為98%,M片段為97%,L片段為98%；與國內(nèi)其他地區(qū)分離株不在同一分支,差異性較大。同時,比對結(jié)果也證實該毒株屬于白蛉病毒屬。 運(yùn)用RT-PCR和分子克隆技術(shù),成功分離位于SFTSV S片段的N蛋白全長基因(738bp),構(gòu)建了克隆載體pMD-19T-SFTSV-NP和原核表達(dá)載體pET-42a(+)-SFTSV-NP,并經(jīng)測序分析。將原核表達(dá)載體pET-42a(+)-SFTSV-NP轉(zhuǎn)化宿主菌BL21(DE3),優(yōu)化了IPTG誘導(dǎo)表達(dá)體系,目的重組蛋白獲得高效表達(dá)。SDS-PAGE電泳分析表明,目的蛋白以包涵體表達(dá)為主。對重組蛋白進(jìn)行鎳柱親和層析,Western blot分析結(jié)果表明,重組蛋白具有良好的抗原性,為SFTSV的早期診斷奠定了生化基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R373.3

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