鸡传贫病毒安徽分离株感染性克隆的构建及其凋亡素抗体的制备

发布时间：2018-04-29 21:45

本文选题：鸡传染性贫血病毒 + 感染性克隆　；参考：《安徽农业大学》2015年硕士论文

【摘要】：鸡传染性贫血病毒(Chicken anemia virus,CIAV)在世界主要养禽国家广泛分布,是危害养禽业的主要病原体之一。鸡是CIAV自然感染的唯一宿主,贫血、淋巴器官萎缩和免疫抑制等是CIAV引起的主要病变,常并发或继发其它病原体,造成经济损失。本研究首先从安徽省某养殖场分离鉴定一株CIAV,并在克隆该株病毒全基因组的基础上构建感染性克隆,表达其毒力因子凋亡素(Apoptin),为进一步研究CIAV致病机理和疫苗设计提供参考。首先,提取安徽某鸡场送检病鸡骨髓中DNA,通过自行设计的3对特异性引物以PCR方法分别扩增病毒3个基因,与pMD18-T载体连接转化大肠杆菌,对阳性克隆进行测序检测。结果表明,病鸡感染了CIAV,将该毒株命名为CIAV-HF211株。通过对CIAV-HF211株与国内外8个分离株的关键结构蛋白VP1基因进行同源进化分析,结果发现基因序列同源性为95%-99.1%,氨基酸序列同源性为96.7%-99.6%,其中与天津分离株AY846844同源性最高。其次,通过设计1对引物(引入EcoR I和Xhol I),扩增出CIAV-HF211株全基因组插入到真核表达载体pcDNA3.1(+)中,构建单拷贝pcD-CIAV。在此基础上,通过另外1对引物扩增第二个全基因组序列并插入到病毒基因组自身携带的BamH I酶切位点处,构建2个CIAV基因组顺次连入一个载体的克隆,命名为pcD-2CIAV。通过腿肌注射1日龄SPF鸡pcD-2CIAV重组质粒(150μg/只),对照组分别注射PBS和病料研磨液,每组注射3-4只。在攻毒后第12d剖检,观察病变并进行病毒基因的检测。结果表明,与PBS对照组相比,pcD-2CIAV重组质粒与CIAV病料感染具有相似的病理变化,且PCR检测结果为阳性,表明了成功构建了1个CIAV感染性克隆。该结果为进一步研究CIAV-HF211株的致病性及研制疫苗奠定了基础。最后,亚克隆CIAV-HF211分离株Apoptin基因,插入原核表达载体pET-32a(+)中,转化大肠杆菌Rosetta(DE3),对阳性菌株经0.2~1.0 mmol/L的IPTG诱导不同时间后经SDS-PAGE电泳检测,并通过镍柱亲和层析纯化His-apoptin融合蛋白,免疫家兔,建立间接ELISA检测Apoptin融合蛋白的抗体水平。以Western blot方法分析融合蛋白反应原性。结果表明,经1.0 mmol/L IPTG诱导18h后可获得较多可溶性表达的apoptin融合蛋白,分子量约为31.6kDa。第三次免疫后,Apoptin融合蛋白的抗血清效价达到1:1280000。Western blot分析表明,该抗血清可与融合蛋白发生特异性反应。综上所述,本研究分离鉴定了来自安徽区域的1株CIAV,并成功构建了该株病毒的感染性克隆,对其Apoptin基因进行了原核表达,建立了间接ELISA方法检测抗Apoptin抗体方法,为进一步研究CIAV致病机理和疫苗的研制奠定了基础。
[Abstract]:Chicken anemia virus (CIAV) is widely distributed in the world and is one of the main pathogens that harm poultry industry. Chicken is the only host of CIAV infection, anemia, lymphoid organ atrophy and immunosuppression are the main pathological changes caused by CIAV, often complicated or secondary to other pathogens, resulting in economic losses. In this study, a strain of CIAV was isolated and identified from a breeding farm in Anhui Province, and an infectious clone was constructed on the basis of cloning the whole genome of the virus, expressing its virulence factor, apoptin, which provides a reference for further study on the pathogenic mechanism of CIAV and the design of vaccine. Firstly, the DNA was extracted from bone marrow of infected chicken in a chicken farm in Anhui province. Three genes of the virus were amplified by PCR method and transformed into Escherichia coli by pMD18-T vector. The positive clones were sequenced. The results showed that the infected chicken was infected with CIAV, and the virus strain was named CIAV-HF211 strain. By homologous evolution analysis of VP1 gene of key structural protein of CIAV-HF211 strain and 8 strains at home and abroad, it was found that the homology of VP1 gene sequence was 95-99.1, the homology of amino acid sequence was 96.7- 99.6um, and the homology was the highest with Tianjin isolate AY846844. Secondly, the whole genome of CIAV-HF211 strain was inserted into eukaryotic expression vector pcDNA3.1 () by designing a pair of primers (EcoR I and Xhol I), and a single copy pcD-CIAV was constructed. On this basis, the second whole genome sequence was amplified by another pair of primers and inserted into the BamH I site carried by the virus genome itself, and two CIAV genomes were sequentially cloned into a vector named pcD-2CIAV. The pcD-2CIAV recombinant plasmid of 1-day-old SPF chicken was injected intramuscularly into the leg, and the control group was injected with PBS and the grinding fluid of diseased materials respectively, 3 to 4 in each group. The lesion was observed and virus gene was detected 12 days after attack. The results showed that the recombinant plasmid pcD-2CIAV had similar pathological changes with CIAV infection compared with PBS control group, and PCR was positive, indicating the successful construction of a CIAV infectious clone. The results laid a foundation for further study on the pathogenicity of CIAV-HF211 strain and the development of vaccine. Finally, the Apoptin gene of CIAV-HF211 isolate was subcloned and inserted into the prokaryotic expression vector pET-32a (), and transformed into E. coli Rosetta-DE3N. The positive strain was induced by 0.21 mmol/L IPTG for different time and then detected by SDS-PAGE electrophoresis. The fusion protein of His-apoptin was purified by nickel column affinity chromatography. To immunize rabbits, indirect ELISA was established to detect the antibody level of Apoptin fusion protein. The reactivity of fusion protein was analyzed by Western blot. The results showed that more soluble apoptin fusion protein could be obtained after 1. 0 mmol/L IPTG induction for 18 h. The molecular weight of the fusion protein was about 31.6 kDa. After the third immunization, the antiserum titer of Apoptin fusion protein reached the level of 1:1280000.Western blot. The results showed that the antiserum could react specifically with the fusion protein. To sum up, a strain of CIAV from Anhui region was isolated and identified, and its infectious clone was successfully constructed, its Apoptin gene was expressed in prokaryotic expression, and indirect ELISA method was established for detection of anti Apoptin antibody. It lays a foundation for further study on the pathogenesis of CIAV and the development of vaccine.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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