表达鸭肝炎病毒VP0蛋白重组鸭瘟病毒的构建
发布时间：2018-08-18 18:09
【摘要】：鸭病毒性肝炎(Duck viral hepatitis,DVH)由鸭肝炎病毒(Duck hepatitis virus,DHV)引起雏鸭发病的烈性传染病,该病潜伏期短、死亡率高,是造成养鸭业经济损失的重要疾病之一鸭病毒性肠炎(Duck viral enteritis,DVE)别名鸭瘟(Duck plague,DP),是由鸭肠炎病毒(Duck enteritis virus,DEV)引起禽类致病的急性败血性传染病,该病主要特点是消化器官受损和实质器官的退行性病变。此病于1957年在我国初次爆发,随后全国大范围的流行对养鸭业造成了极为严重的经济损失。本研究以实验室保存的pET30a载体,针对DHAV-1 VP0基因进行设计引物并对VP0基因进行扩增,成功构建pET30a-VP0载体并进行蛋白的表达及纯化。纯化后的蛋白免疫新西兰大耳兔,3次免疫后采血进行免疫效价的测定,成功制备VP0蛋白兔多抗血清。运用实验室成功构建的转染粘粒拯救鸭瘟病毒系统,选用实验室保存的pCAGGS-VP0经过BP反应构建成为pDONR-PCA-VP0,再经过LR反应把PCA-VP0表达盒重组到粘粒2上命名为2-UL41-VP0,2-UL41-VP0代替2粘粒拯救表达VP0蛋白的重组病毒rDEV-VP0。rDEV-VP0在CEF上进行转染细胞经过培养传代后提取病毒DNA进行PCR鉴定,成功构建重组病毒。选取60羽雏鸭分组进行动物免疫效果评价试验,分为rDEV-VP0免疫2组,DEV攻毒对照组,DHAV攻毒对照组,C-KCE免疫对照组及阴性对照组。免疫四天进行DEV和DHAV强毒的攻击。结果表明rDEV-VP0免疫雏鸭4天后可对CSC DEV的攻击可以达到完全的免疫保护,对DHV 161/79的攻击可提供70%的免疫保护。
[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.
【学位授予单位】：牡丹江师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：牡丹江师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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