中蜂囊状幼虫病毒基因分型及其代表毒株生物学特性比较

发布时间：2018-02-15 05:59

  本文关键词： 中华蜜蜂囊状幼虫病毒 分子生物学特性 致病性 理化特性 免疫原性　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的通过对收集于2008年-2015年我国部分地区疑似感染中华蜜蜂囊状幼虫病毒(Chinese sacbrood bee virus,CSBV)的幼虫进行检测,并对CSBV新分离株进行筛选命名。以CSBV结构蛋白基因VP1为靶基因,对CSBV进行基因分型并对各基因型的代表毒株进行筛选,在此基础上,对各个代表株进行分子生物学特性、致病性、理化特性以及免疫原性进行比较分析,为CSBV病毒的生物学特性研究及其抗CSBV生物制剂研发奠定基础。方法通过已建立的RT-PCR方法对本实验室保存的2008年-2015年我国部分地区疑似感染CSBV病料进行分离鉴定,对来自同一地区、同一时期,同源性达到99.9%以上的阳性样品,作为一个毒株进行命名。以CSBV结构蛋白VP1基因为靶基因,对分离到得CSBV毒株和Gen Bank上公布的CSBV毒株,进行遗传进化分析,并根据遗传进化分析结果确定CSBV基因型,筛选各基因型的代表毒株。对各基因型代表毒株进行全基因组克隆,并对其核苷酸序列及其推导氨基酸序列、遗传背景等分子生物学特性进行分析。在此基础上,分别接种2日龄中华蜜蜂幼虫进行本体动物感染试验,通过观察幼虫死亡率、临床症状及病理组织变化,比较代表毒株的致病性,同时对三株病毒的温度、p H值和有机溶剂等理化指标的敏感性进行研究,并进行交叉免疫保护性试验比较分析。根据对分离到16株CSBV和Gen Bank上公布的7株CSBV遗传进化分析结果,将CSBV分为三个基因型,分别在三个基因型中选择LNQY-2008(2008年分离自辽宁清源),SXYL-2015(2015年分离自陕西榆林)和JLCBS-2014(2014年分离自吉林长白山),作为代表毒株进行研究。LNQY-2008、SXYL-2015和JLCBS-2014分子生物学特性比较分析结果表明,与SXYL-2015株相比较,LNQY-2008株在282 291和299 301氨基酸位置上分别存在10个氨基酸和3个氨基酸的缺失,JLCBS-2014株在284 300处有17个氨基酸缺失,且3株CSBV内的解旋酶,蛋白酶以及Rd Rp的保守结构域与其他哺乳类和昆虫类动物的小核糖核酸病毒相似。幼虫感染试验结果表明,三株CSBV感染幼虫后临床症状相似,病理变化基本相同且幼虫感染后死亡率差异不显著(P0.05)。通过对理化性质研究表明,三株CSBV均对酸性条件具有很高的稳定性,在p H=3.0的条件下仍能存活;对乙醚和氯仿等有机溶剂具有极强的抵抗力;耐受最高温度最高达75℃,75℃及其以上时CSBV易失活。交叉免疫保护试验表明,三株CSBV间存在交叉免疫反应。结果结论1、基于VP1基因遗传进化分析发现16株分离株CSBV以及Gen Bank上公布的7株CSBV可以分类为3个基因型。2、全基因组序列对比分析3个CSBV代表性基因型毒株发现,与毒株SXYL-2015相较,LNQY-2008株和JLCBS-2014株均出现了不同程度的编码基因及氨基酸的缺失。3、在致病性、理化特性和免疫交叉保护性进行比较分析结果表明LNQY-2008、SXYL-2015和JLCBS-2014株的CSBV致病性和理化性质差异不显著(P0.05),且存在免疫交叉保护。
[Abstract]:Objective to detect the larvae of Chinese sacbrood bee virus (CSBV) collected from 2008 to 2015 in some parts of China, and to screen and name the new CSBV isolate. The CSBV structural protein gene VP1 was used as the target gene. On the basis of the genotyping of CSBV and the screening of the representative strains of each genotype, the molecular biological characteristics, pathogenicity, physicochemical properties and immunogenicity of each representative strain were compared and analyzed. Methods the biological characteristics of CSBV virus and the research and development of CSBV resistant biologics were studied. Methods the samples of suspected CSBV infection from 2008 to 2015 in our laboratory were isolated and identified by using the established RT-PCR method. The positive samples with homology of more than 99.9% from the same region and the same period were named as a virus strain. Using the VP1 gene of CSBV structural protein as the target gene, the CSBV virus strain and the CSBV virus strain published on Gen Bank were isolated. The genetic evolution analysis was carried out. According to the result of genetic evolution analysis, the CSBV genotype was determined, the representative strains of each genotype were screened, the whole genome of the representative strains were cloned, and the nucleotide sequence and amino acid sequence were deduced. On the basis of the analysis of molecular biological characteristics such as genetic background, two day old Chinese honeybee larvae were inoculated to carry out the infection test in vivo. The mortality rate, clinical symptoms and pathological changes of the larvae were observed. The pathogenicity of the three strains was compared, and the sensitivity of temperature pH and organic solvents were studied. According to the results of genetic evolution analysis of 7 CSBV strains isolated from 16 CSBV and Gen Bank, CSBV was divided into three genotypes. LNQY-2008 (isolated from SXYL-2015, Liaoning Province on 2008) and JLCBS-2014 (isolated from Yulin, Shaanxi Province on 2015) and JLCBS-2014 (isolated from Jilin Province, Jilin Province) were selected from three genotypes, respectively. The molecular biological characteristics of SXYL-2015 and JLCBS-2014 were studied as representative strains. Compared with the SXYL-2015 strain, there were 10 amino acids and 3 amino acid deletions at the amino acid position of 282n291 and 299N301, respectively. The JLCBS-2014 strain had 17 amino acid deletions at 284,300, and three CSBV helicases. The conserved domains of protease and Rd RP were similar to those of small ribonucleic acid viruses in other mammals and insects. The results of larval infection showed that the clinical symptoms of the three strains of CSBV were similar to those of the larvae. The pathological changes were basically the same and there was no significant difference in larval mortality after infection (P 0.05). The physicochemical properties of the three strains of CSBV were studied. The results showed that the three strains of CSBV had high stability to acidic conditions and could still survive under pH 3.0. It has strong resistance to organic solvents such as ether and chloroform, and CSBV is easily inactivated when the highest temperature is 75 鈩，

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