dhav3 ls株遗传变异分析 vp1蛋白表达及荧光定量pcr型方法的建立.docx

优秀毕业论文 精品参考文献资料 位氨基酸区段的可能性最大，同时这些位点也是最易变异的位点。针对高 位氨基酸区段的可能性最大，同时这些位点也是最易变异的位点。针对高 变区选取的第132．240位氨基酸区段得到成功表达，纯化的融合蛋白分子量 约为38ku。Western blot分析结果表明，融合蛋白能被抗GST标签鼠单克 隆抗体特异性识别，证明该蛋白具有良好的反应原性。 本研究还针对1、3型鸭甲肝病毒5端保守区设计一对特异性引物，采 用SYBR Green I荧光染料，成功建立了一种基于荧光定量PCR熔解曲线 法的DHAV分型检测方法。结果表明，建立的方法特异、快速、敏感，对 1．0x102～1．0x109 copies／I．tL浓度的模板均有较好的检测效果，最低检出量均 为100 copies4tL，是普通PCR的100倍；特异性好，排除常见禽源病毒的 干扰；对高、中、低不同浓度质粒标准品检测表现出良好重复性。利用建 立方法对DHAV-1和DHAV-3感染雏鸭后在体内组织器官的增殖规律进 行探究，发现鸭甲型肝炎病毒广泛增殖于鸭的大部分组织脏器内。两种病 毒在组织器官中的增殖规律大体一致，其中对肝脏最具有嗜性，脾脏、肾 脏、肺脏中病毒滴度也很高，进而从病毒含量角度解释了DHAV感染雏鸭 后在肝脏、脾脏、肾脏、肺脏等主要靶器官引起的特征病变。 关键词：鸭甲肝病毒分离鉴定序列分析结构蛋白VPl原核表达荧光 定量PCR 万方数据 GENETIC GENETIC VARIATION ANALYSIS AND VP l PROTEIN EXPRESSION OF DHAV_3 LS STRAIN AND ESTABLISH姬NT OF FQ—PCR GENOTYPING METHOD A。B STRACT Duck viral hepatitis(DVH)is known as an acute，highly lethal viral disease within 3 weeks old ducklings by duck hepatitis virus，seriously affecting the development of duck industry．At present，the main pathogen of DHV is the duck hepatitis A virus(DHAV)in China．Therefore，it is significant to conduct studies of DHAV isolation，genetic variation，main structural protein expression and differential diagnosis of pathogen for future research on DHAV molecular epidemiology,pathogen detection and disease prevention and contr01． A strain of virus，named as LS strain，was isolated and identified by duck embryo proliferation，animal regression test，ELD50 and RT-PCR method from 1 case of suspected DVH．The complete genome sequences of the isolate were amplified，cloned and analyzed．The result showed that the whole genome of LS strain was 7 8 1 5 nt in length excluding the cap structure，including 5’UTR(652 nt)，ORF(6 75 6nt)and 3 UTR(3 66nt)back with at least 26A poly(A)tail LS strain shared the highest nucleotide and amino acid homology with DHAV-3 reference strains reaching 97．4％and 99％；furthermore，LS strain and DHAV-3 C-BLZ，SD 1 20 1 strain were in the same genotype，and they might come from the same ancestor．As a result LS strain could