發(fā)布時(shí)間：2018-03-08 19:48

  本文選題：新城疫病毒　切入點(diǎn)：耐熱株　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：新城疫病毒(Newcastle disease virus,NDV)是嚴(yán)重危害養(yǎng)禽業(yè)的烈性傳染病——新城疫(Newcastle disease,ND)的病原。由于V4和1-2等耐熱ND弱毒疫苗的貯存和運(yùn)輸不需要依賴?yán)滏?在熱帶、亞熱帶國家和地區(qū)已經(jīng)得到廣泛應(yīng)用。我國上世紀(jì)90年代引進(jìn)了 NDV耐熱毒株V4,但由于知識(shí)產(chǎn)權(quán)等的限制,至今未大規(guī)模應(yīng)用于生產(chǎn)。本研究從保存的40多株NDV中篩選到1個(gè)耐熱強(qiáng)毒株(HR09株);測定了該毒株的全基因組序列,并分析了其分子生物學(xué)特性;建立了該毒株的反向遺傳操作系統(tǒng),并對其F基因進(jìn)行突變,得到NDV耐熱弱毒株。本研究為進(jìn)一步揭示NDV耐熱毒株的耐熱機(jī)制和創(chuàng)制耐熱ND弱毒疫苗奠定了基礎(chǔ)。1新城疫病毒耐熱株HR09鑒定及全基因組序列測定將本室保存的40多個(gè)NDV分離株與V4、LaSota等參考毒株共同進(jìn)行耐熱性試驗(yàn)。以56℃耐熱處理30 min時(shí)仍具有血凝性作為篩選標(biāo)準(zhǔn),獲得1個(gè)耐熱毒株——HR09株。HR09株經(jīng)蝕斑純化后進(jìn)行生物學(xué)特性鑒定,顯示該毒株的MDT為57.6 h,ICPI為1.8,IVPI為1.56,符合NDV強(qiáng)毒株特征。設(shè)計(jì)10對引物,對HR09株全基因組序列進(jìn)行擴(kuò)增、測定和生物信息學(xué)分析,結(jié)果顯示,該毒株基因組全長為15192 nt,推導(dǎo)的F蛋白裂解位點(diǎn)氨基酸序列為112RRQKRF117,HN蛋白長度為571個(gè)氨基酸殘基,符合NDV強(qiáng)毒株特征。HR09株基因組GC含量為46.27%,與耐熱的V4株、1-2株全基因組的核苷酸序列同源性為86.7%和85.4%,6個(gè)結(jié)構(gòu)蛋白編碼基因的核苷酸序列同源性介于83.1%～95.6%之間、推導(dǎo)的氨基酸序列同源性介于81.6%～98.0%之間。基于F基因全長所構(gòu)建的進(jìn)化樹顯示,HR09株屬于基因ⅷ型毒株。2新城疫病毒耐熱株HR09反向遺傳操作系統(tǒng)的構(gòu)建設(shè)計(jì)了多對引物,構(gòu)建了 HR09株基因組的全長cDNA克隆以及含有該毒株NP、P和L基因的3個(gè)真核表達(dá)質(zhì)粒,共轉(zhuǎn)染BSR-T7/5細(xì)胞,拯救出病毒NDV/rHR09。對獲救病毒的耐熱性進(jìn)行測定,顯示其經(jīng)56℃處理60 min時(shí)仍具有血凝活性,說明NDV/rHR09株與親本株的耐熱性相似。NDV/rHR09株的MDT值為60 h,ICPI值為1.625,仍為NDV強(qiáng)毒株。3新城疫病毒耐熱弱毒株rAHR09的構(gòu)建設(shè)計(jì)兩對引物,經(jīng)Overlap PCR的方法將NDV/rHR09毒株F基因裂解位點(diǎn)的112、115和117位堿性氨基酸突變成弱毒株特征的非堿性氨基酸,構(gòu)建了轉(zhuǎn)錄載體prNDV/HR09,將轉(zhuǎn)錄載體與NP、P和L基因的真核表達(dá)質(zhì)粒共轉(zhuǎn)染BSR-T7/5細(xì)胞,拯救出病毒NDV/rAHR09。經(jīng)56。C、45min處理,NDV/rAHR09株仍具有血凝性,MDT值120h,ICPI值為0.057。結(jié)果表明,NDV/rAHR09株具有耐熱性,且毒力已明顯致弱,可以作為耐熱ND弱毒疫苗的候選毒株。
[Abstract]:Newcastle disease virus (NDV) is the pathogen of Newcastle disease (NDV), a severe infectious disease in poultry industry. Because the storage and transportation of heat-resistant NDV vaccines such as V4 and 1-2 do not depend on cold chains, they are in the tropics. Subtropical countries and regions have been widely used. In -10s, China introduced NDV heat-resistant strain V4, but due to the limitations of intellectual property rights, Up to now, no large scale application has been made. In this study, a heat-resistant and virulent strain HR09 was screened out of more than 40 strains of NDV preserved, the whole genome sequence of the strain was determined, and its molecular biological characteristics were analyzed. The reverse genetic operating system of the strain was established, and the F gene was mutated. NDV heat-resistant attenuated strain was obtained. This study laid a foundation for further revealing the heat-resistant mechanism of NDV heat-resistant strain and creating a heat-resistant ND attenuated vaccine. 1. The Newcastle disease virus heat-resistant strain was identified by HR09 and the whole genome sequence was sequenced. More than 40 NDV isolates were tested for heat resistance with reference strains V4 and LaSota. Hemagglutination at 56 鈩，

本文編號(hào)：1585305