小反刍兽疫病毒FY株分离、N基因表达及全基因组序列解析

发布时间：2018-03-16 09:36

本文选题：小反刍兽疫　切入点：病毒分离　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：小反刍兽疫(Pestedes petits ruminants,PPR)主要发生在发展中国家,严重危害养羊业的发展,其跨境传播能力强,致死率最高可达100%。我国目前报道的小反刍兽疫病毒(Pestedes petits ruminants virus,PPRV)流行毒株主要集中于西藏和新疆等养羊业比较集中的地区,其它地区的流行毒株少有报道。本研究对2014年从浙江富阳分离的一株PPRV流行毒株(FY株)进行了分离和鉴定,同时对其全基因组进行了测序和分析,以期为我国PPRV分子流行病学研究提供新的参考资料,并丰富PPRV基因库,这将有助于分析病毒变异状况,研究PPRV在我国的遗传背景等信息,选择适合的疫苗以及研发新型疫苗。本论文内容包括以下3个方面。1、PPRV FY株的分离和鉴定:将从浙江省富阳市采集的PPRV病料进行研磨,经离心、除菌后,分别接种于长满单层的Vero细胞、BHK细胞及CV细胞(CV)。培养96h后,收集细胞培养物,再在上述细胞中盲传3-5代。结果发现PPRV FY株在vero细胞和BHK细胞中均没有明显细胞病变出现,但是在CV细胞中出现典型细胞病变,主要表现为:细胞变圆、脱落、出现合胞体。待细胞出现80%左右病变时,收获细胞培养物,然后分别使用RT-PCR、Western Blot、间接免疫荧光及电镜负染等方法对病毒分离物进行鉴定。检测结果表明,从CV细胞培养物中能特异性地检测到PPRV的基因组,Western Blot、间接免疫荧光检测结果均证明用CV细胞分离到的病毒可与PPRV单抗F发生特异性反应。将收获的CV细胞培养物进行超速离心,纯化出病毒,然后用电子显微镜进行观察,可以看到具有囊膜的,直径在150-300nm之间的病毒颗粒,符合已报道的PPRV病毒颗粒形态学特征。上述研究结果证明,我们成功应用CV细胞分离到了PPRV,并将其命名为FY株。2、PPRV FY株N基因的原核表达及多抗的制备:扩增PPRV FY株N基因序列,连接到pET-32a载体上测序分析,测序正确后转化到(E.coli)BL21(DE3)菌株中,IPTG诱导,表达重组N蛋白,并采用包涵体纯化的方法纯化重组蛋白,将纯化蛋白免疫实验兔,制备多克隆抗体,并使用WesternBlot及间接免疫荧光等方法对得到的多抗进行鉴定,结果表明所制备多抗特异性良好并可用于PPRV的初步检测。3、PPRV FY株的全基因组序列测定及分析:根据已经发表的PPRV参考毒株序列,设计了 11对特异性扩增引物,然后用RT-PCR方法分段扩增出PPRV FY株的全基因组序列,应用DNAstar软件和MEGA软件对PPRV FY株及参考毒株的基因序列进行了比对和分析,并根据N基因核苷酸序列绘制了系统进化树。试验结果表明PPRVFY株基因组全长为15948nt,推测编码6种结构蛋白(N、P、L、M、F和H),2种非结构蛋白(C和V),PPRV各毒株转录起始序列和转录终止序列都高度保守。同源性比较分析结果显示,PPRVFY株与科特迪瓦来源毒株具有最低的同源性,然而与西藏来源毒株具有最高同源性,其基因间隔区N-P长度一致,相似度最高,而M-F相似度最低,通过比较PPRVFY株与参考毒株基因组末端调控序列,还可以看出在整个麻疹病毒属GP区和AGP区保守性较高,且存在不同程度的互补。氨基酸序列分析结果揭示PPRVFY株在保守区域出现21处氨基酸突变,这些突变对其所编码蛋白的结构和功能有何影响尚需要进一步研究。根据PPRV N基因序列,我们对PPRV FY株及参考毒株进行了系统进化分析。分析结果发现,PPRVFY株与其它亚洲源PPRV野毒株共同形成一个拓扑群,属于第IV谱系。综上所述,本研究成功分离到了 2014年流行于浙江省的PPRV流行毒株FY株,该毒株对细胞的适应性不强,仅能在表达其受体基因的细胞系中增殖。在此基础上,我们完成了对其全基因组序列的测定、N基因的原核表达及生物信息学分析,证明PPRVFY株属于基因IV型,并且与疫苗株的同源性较低,但与我国报道的其它野毒株具有较高同源性。
[Abstract]:Peste des petits ruminants (Pestedes petits ruminants, PPR) occurs mainly in developing countries, serious harm to the development of sheep industry and the cross-border transmission capacity, the highest mortality rate of up to 100%. in China currently reported PPRV (Pestedes petits ruminants virus, PPRV) strains are mainly concentrated in the Tibet and Xinjiang sheep the industry is relatively concentrated areas, other areas of the epidemic strains are rarely reported. The study on 2014 Fuyang strain isolated from Zhejiang PPRV strains (FY strain) were isolated and identified, and the whole genome was sequenced and analyzed, in order to provide reference for the study of new China PPRV molecular epidemiology PPRV, and enrich the gene pool, which will be helpful to analyze the virus mutation status, research in China PPRV genetic background information, select the appropriate vaccine and develop new vaccines. The content of this thesis includes the following 3 aspects of.1, isolation and identification of PPRV FY strain: from Zhejiang city of Fuyang province collected PPRV samples were ground by centrifugation, after sterilization, were inoculated into full monolayer Vero cells, BHK cells and CV cells (CV). After 96h culture, collecting cell cultures, and then in the above cells in 3-5 blind passages. The results showed that PPRV FY strain in Vero cells and BHK cells were no obvious cell lesions, but in CV cells showed typical cell pathological changes, the main performance is: the cells became round, shedding appears syncytial cells appeared to be. 80% lesions, harvest cell culture then, using RT-PCR, Western Blot, indirect immunofluorescence and electron microscopy negative staining method for identification of isolates of the virus. The detection results show that the compound can specifically detect PPRV genome, Western Blot from CV cell culture, indirect immunofluorescence test results showed that with CV cells The isolated virus with PPRV monoclonal antibody F specific reaction. After CV cell culture by centrifugation, and purified virus, and then was examined by electron microscopy, can be seen with the capsule, the diameter between 150-300nm particles with morphological features of PPRV virus particles have been reported the results of the study. We proved that the successful application of CV cells to PPRV, and named FY strain.2, prokaryotic expression and polyclonal antibody of N gene of PPRV strain FY preparation: FY amplification of PPRV N gene sequence analysis, connected to pET-32a vector and sequencing, sequencing and transformed into (E.coli) BL21 (DE3) IPTG induced strains, and expression of recombinant N protein, and the method of purification of inclusion body of recombinant protein, the purified protein immunized rabbit, polyclonal antibody, and using WesternBlot and indirect immunofluorescence method to obtain the polyclonal antibody into For identification, the results show that the preparation of the preliminary detection of.3 antibody and PPRV can be used for the determination and analysis of the complete genome sequence of PPRV strain FY PPRV reference strains: according to the published sequence, designed 11 pairs of primers, and then use the RT-PCR method to expand the segmented genome of PPRV FY strain the application of DNAstar software and MEGA software of PPRV FY strains and reference strains of the gene sequence was compared and analyzed, and draw the phylogenetic tree based on the nucleotide sequence of N gene. The experimental results showed that the full-length genome of PPRVFY strain was 15948nt, speculated that the 6 structural proteins (N, P, encoding L, M, F and H 2), non structural proteins (C and V), PPRV strains transcription initiation sequence and transcription termination sequences are highly conserved. Homology analysis showed that PPRVFY strain and strain with the lowest homology from Ivory Coast, but with Tibet source of poison Strains with the highest homology, the length of the intergenic region N-P and M-F, the highest similarity, the lowest similarity, through the comparison of the PPRVFY strains and the reference strains genome end regulatory sequences can also be seen in the measlesvirus GP conservation area and AGP area is high, and does not have the same degree of complementary amino acid sequence analysis revealed PPRVFY. In 21 strains of conserved regions of amino acid mutations, these mutations on the structure and function of the protein encoding impact still needs further research. According to the PPRV sequence of N gene, we performed phylogenetic analysis of PPRV FY strains and reference strains. The results showed that PPRVFY strain and other Asian wild strains together form the source PPRV a topological group belongs to the IV lineage. In conclusion, PPRV strains FY strains in this study successfully isolated the 2014 popular in Zhejiang Province, the strain on cell compatibility is not Strong proliferation only in its receptor gene expression in cell lines. On this basis, we have completed the determination of the whole genome sequence analysis, prokaryotic expression and biological information of N gene, that PPRVFY belonged to genotype IV, and vaccine strains with low homology, but other wild strains reported in China and has a high homology.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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