表達鴨肝炎病毒VP0蛋白重組鴨瘟病毒的構(gòu)建
發(fā)布時間：2018-08-18 18:09
【摘要】：鴨病毒性肝炎(Duck viral hepatitis,DVH)由鴨肝炎病毒(Duck hepatitis virus,DHV)引起雛鴨發(fā)病的烈性傳染病,該病潛伏期短、死亡率高,是造成養(yǎng)鴨業(yè)經(jīng)濟損失的重要疾病之一鴨病毒性腸炎(Duck viral enteritis,DVE)別名鴨瘟(Duck plague,DP),是由鴨腸炎病毒(Duck enteritis virus,DEV)引起禽類致病的急性敗血性傳染病,該病主要特點是消化器官受損和實質(zhì)器官的退行性病變。此病于1957年在我國初次爆發(fā),隨后全國大范圍的流行對養(yǎng)鴨業(yè)造成了極為嚴重的經(jīng)濟損失。本研究以實驗室保存的pET30a載體,針對DHAV-1 VP0基因進行設計引物并對VP0基因進行擴增,成功構(gòu)建pET30a-VP0載體并進行蛋白的表達及純化。純化后的蛋白免疫新西蘭大耳兔,3次免疫后采血進行免疫效價的測定,成功制備VP0蛋白兔多抗血清。運用實驗室成功構(gòu)建的轉(zhuǎn)染粘粒拯救鴨瘟病毒系統(tǒng),選用實驗室保存的pCAGGS-VP0經(jīng)過BP反應構(gòu)建成為pDONR-PCA-VP0,再經(jīng)過LR反應把PCA-VP0表達盒重組到粘粒2上命名為2-UL41-VP0,2-UL41-VP0代替2粘粒拯救表達VP0蛋白的重組病毒rDEV-VP0。rDEV-VP0在CEF上進行轉(zhuǎn)染細胞經(jīng)過培養(yǎng)傳代后提取病毒DNA進行PCR鑒定,成功構(gòu)建重組病毒。選取60羽雛鴨分組進行動物免疫效果評價試驗,分為rDEV-VP0免疫2組,DEV攻毒對照組,DHAV攻毒對照組,C-KCE免疫對照組及陰性對照組。免疫四天進行DEV和DHAV強毒的攻擊。結(jié)果表明rDEV-VP0免疫雛鴨4天后可對CSC DEV的攻擊可以達到完全的免疫保護,對DHV 161/79的攻擊可提供70%的免疫保護。
[Abstract]:Duck viral hepatitis (Duck viral hepatitis (DHV) caused by duck hepatitis virus (Duck hepatitis virus (DHV) caused a strong infectious disease in ducklings. The incubation period of the disease was short and the mortality rate was high. Duck plague (Duck plagueae DP), one of the most important diseases causing economic loss in duck breeding, is an acute septic infectious disease caused by duck enteritis virus (Duck enteritis virus), which is caused by duck enteritis virus (Duck enteritis virus). The disease is characterized by damage to digestive organs and degenerative lesions of parenchymal organs. The disease broke out for the first time in China in 1957, and then it caused a serious economic loss to the duck industry in the whole country. In this study, we designed primers for DHAV-1 VP0 gene and amplified VP0 gene with pET30a vector preserved in laboratory. We successfully constructed pET30a-VP0 vector and expressed and purified the protein. After the purified protein was immunized with New Zealand big ear rabbit, the immune titer was determined after three immunizations, and the polyantiserum of VP0 protein was prepared successfully. Using the successfully constructed lab transfection mucus to save duck plague virus system, After BP reaction, the pCAGGS-VP0 stored in the laboratory was constructed into pDONR-PCA-VP0, and then the PCA-VP0 expression box was recombined into clay 2 by LR reaction. Instead of 2-UL41-VP0, 2-UL41-VP0 was replaced by 2-UL41-VP0 to save the recombinant virus rDEV-VP0.rDEV-VP0 expressing VP0 protein. After transfection on CEF, the recombinant virus rDEV-VP0.rDEV-VP0 expressing VP0 protein was transformed into the cells transfected with pDONR-PCA-VP0. After culture and passage, the virus DNA was extracted for PCR identification. The recombinant virus was successfully constructed. A total of 60 ducklings were divided into two groups: rDEV-VP0 immunization group (n = 2) and control group (C KCE control group) and negative control group (n = 10). DEV and DHAV were immunized for four days. The results showed that rDEV-VP0 immunized ducklings could achieve complete immune protection against CSC DEV 4 days later, and 70% of DHV 161 / 79 attack could provide immunity protection.
【學位授予單位】：牡丹江師范學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
[Abstract]:Duck viral hepatitis (Duck viral hepatitis (DHV) caused by duck hepatitis virus (Duck hepatitis virus (DHV) caused a strong infectious disease in ducklings. The incubation period of the disease was short and the mortality rate was high. Duck plague (Duck plagueae DP), one of the most important diseases causing economic loss in duck breeding, is an acute septic infectious disease caused by duck enteritis virus (Duck enteritis virus), which is caused by duck enteritis virus (Duck enteritis virus). The disease is characterized by damage to digestive organs and degenerative lesions of parenchymal organs. The disease broke out for the first time in China in 1957, and then it caused a serious economic loss to the duck industry in the whole country. In this study, we designed primers for DHAV-1 VP0 gene and amplified VP0 gene with pET30a vector preserved in laboratory. We successfully constructed pET30a-VP0 vector and expressed and purified the protein. After the purified protein was immunized with New Zealand big ear rabbit, the immune titer was determined after three immunizations, and the polyantiserum of VP0 protein was prepared successfully. Using the successfully constructed lab transfection mucus to save duck plague virus system, After BP reaction, the pCAGGS-VP0 stored in the laboratory was constructed into pDONR-PCA-VP0, and then the PCA-VP0 expression box was recombined into clay 2 by LR reaction. Instead of 2-UL41-VP0, 2-UL41-VP0 was replaced by 2-UL41-VP0 to save the recombinant virus rDEV-VP0.rDEV-VP0 expressing VP0 protein. After transfection on CEF, the recombinant virus rDEV-VP0.rDEV-VP0 expressing VP0 protein was transformed into the cells transfected with pDONR-PCA-VP0. After culture and passage, the virus DNA was extracted for PCR identification. The recombinant virus was successfully constructed. A total of 60 ducklings were divided into two groups: rDEV-VP0 immunization group (n = 2) and control group (C KCE control group) and negative control group (n = 10). DEV and DHAV were immunized for four days. The results showed that rDEV-VP0 immunized ducklings could achieve complete immune protection against CSC DEV 4 days later, and 70% of DHV 161 / 79 attack could provide immunity protection.
【學位授予單位】：牡丹江師范學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
【參考文獻】
相關期刊論文 前10條
1 張苗苗;李慧昕;韓宗璽;劉懷然;王玉龍;邵昱昊;孔憲剛;;表達新城疫病毒F基因重組鴨腸炎病毒的構(gòu)建[J];中國預防獸醫(yī)學報;2016年02期
2 王安平;朱善元;吳雙;王永娟;左偉勇;洪偉鳴;;鴨新城疫病毒M基因的原核表達與多抗制備[J];河南農(nóng)業(yè)科學;2016年01期
3 王安平;朱善元;王永娟;;鴨甲型肝炎病毒1型3C基因的原核表達與多抗的制備[J];江蘇農(nóng)業(yè)科學;2015年08期
4 李奇蒙;陳普成;柳金雄;姜永萍;王笑梅;田雨;胡永浩;陳化蘭;;表達傳染性法氏囊病毒VP2蛋白的重組火雞皰疹病毒的構(gòu)建[J];中國預防獸醫(yī)學報;2014年07期
5 王衡;劉博奇;任曉峰;寧章勇;;豬氨肽酶N多抗血清的制備及活性分析[J];中國獸醫(yī)科學;2011年08期
6 趙偉;李傳峰;陳宗艷;劉光清;;Ⅰ型鴨肝炎病毒內(nèi)部核糖體進入位點的結(jié)構(gòu)與功能研究[J];中國預防獸醫(yī)學報;2011年01期
7 曾巖;任曉峰;蔡皓t，
本文編號：2190273
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2190273.html
最近更新
教材專著
