本文關(guān)鍵詞： 中華蜜蜂囊狀幼蟲病毒 分子生物學(xué)特性 致病性 理化特性 免疫原性　出處：《錦州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的通過對收集于2008年-2015年我國部分地區(qū)疑似感染中華蜜蜂囊狀幼蟲病毒(Chinese sacbrood bee virus,CSBV)的幼蟲進(jìn)行檢測,并對CSBV新分離株進(jìn)行篩選命名。以CSBV結(jié)構(gòu)蛋白基因VP1為靶基因,對CSBV進(jìn)行基因分型并對各基因型的代表毒株進(jìn)行篩選,在此基礎(chǔ)上,對各個代表株進(jìn)行分子生物學(xué)特性、致病性、理化特性以及免疫原性進(jìn)行比較分析,為CSBV病毒的生物學(xué)特性研究及其抗CSBV生物制劑研發(fā)奠定基礎(chǔ)。方法通過已建立的RT-PCR方法對本實驗室保存的2008年-2015年我國部分地區(qū)疑似感染CSBV病料進(jìn)行分離鑒定,對來自同一地區(qū)、同一時期,同源性達(dá)到99.9%以上的陽性樣品,作為一個毒株進(jìn)行命名。以CSBV結(jié)構(gòu)蛋白VP1基因為靶基因,對分離到得CSBV毒株和Gen Bank上公布的CSBV毒株,進(jìn)行遺傳進(jìn)化分析,并根據(jù)遺傳進(jìn)化分析結(jié)果確定CSBV基因型,篩選各基因型的代表毒株。對各基因型代表毒株進(jìn)行全基因組克隆,并對其核苷酸序列及其推導(dǎo)氨基酸序列、遺傳背景等分子生物學(xué)特性進(jìn)行分析。在此基礎(chǔ)上,分別接種2日齡中華蜜蜂幼蟲進(jìn)行本體動物感染試驗,通過觀察幼蟲死亡率、臨床癥狀及病理組織變化,比較代表毒株的致病性,同時對三株病毒的溫度、p H值和有機(jī)溶劑等理化指標(biāo)的敏感性進(jìn)行研究,并進(jìn)行交叉免疫保護(hù)性試驗比較分析。根據(jù)對分離到16株CSBV和Gen Bank上公布的7株CSBV遺傳進(jìn)化分析結(jié)果,將CSBV分為三個基因型,分別在三個基因型中選擇LNQY-2008(2008年分離自遼寧清源),SXYL-2015(2015年分離自陜西榆林)和JLCBS-2014(2014年分離自吉林長白山),作為代表毒株進(jìn)行研究。LNQY-2008、SXYL-2015和JLCBS-2014分子生物學(xué)特性比較分析結(jié)果表明,與SXYL-2015株相比較,LNQY-2008株在282 291和299 301氨基酸位置上分別存在10個氨基酸和3個氨基酸的缺失,JLCBS-2014株在284 300處有17個氨基酸缺失,且3株CSBV內(nèi)的解旋酶,蛋白酶以及Rd Rp的保守結(jié)構(gòu)域與其他哺乳類和昆蟲類動物的小核糖核酸病毒相似。幼蟲感染試驗結(jié)果表明,三株CSBV感染幼蟲后臨床癥狀相似,病理變化基本相同且幼蟲感染后死亡率差異不顯著(P0.05)。通過對理化性質(zhì)研究表明,三株CSBV均對酸性條件具有很高的穩(wěn)定性,在p H=3.0的條件下仍能存活;對乙醚和氯仿等有機(jī)溶劑具有極強(qiáng)的抵抗力;耐受最高溫度最高達(dá)75℃,75℃及其以上時CSBV易失活。交叉免疫保護(hù)試驗表明,三株CSBV間存在交叉免疫反應(yīng)。結(jié)果結(jié)論1、基于VP1基因遺傳進(jìn)化分析發(fā)現(xiàn)16株分離株CSBV以及Gen Bank上公布的7株CSBV可以分類為3個基因型。2、全基因組序列對比分析3個CSBV代表性基因型毒株發(fā)現(xiàn),與毒株SXYL-2015相較,LNQY-2008株和JLCBS-2014株均出現(xiàn)了不同程度的編碼基因及氨基酸的缺失。3、在致病性、理化特性和免疫交叉保護(hù)性進(jìn)行比較分析結(jié)果表明LNQY-2008、SXYL-2015和JLCBS-2014株的CSBV致病性和理化性質(zhì)差異不顯著(P0.05),且存在免疫交叉保護(hù)。
[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，

本文編號：1512589