犬传染性肝炎DNA疫苗的构建和免疫原性研究
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摘要
核酸疫苗是90年代兴起的新型疫苗,已在多种疾病的防治中显示出巨大的潜能。新一代DNA疫苗能全面的诱发免疫反应,且生产易于标准化,从而得到人们的重视。随着核酸疫苗研究的不断深入,一个又一个预防动物传染性疾病的核酸疫苗将应运而生。本实验针对犬传染性疾病预防需要新型核酸疫苗这一趋势,进行了如下研究:①构建预防犬传染性肝炎的核酸疫苗pVAX1-CpG-Loop,并对其免疫效果进行评价;②构建原核蛋白表达质粒pET28a-Loop,表达、纯化Loop蛋白;③制备鼠抗Loop蛋白单克隆抗体并对克隆细胞株进行鉴定。
犬传染性肝炎是由犬传染性肝炎病毒感染引起。犬传染性肝炎病毒又称犬腺病毒(Canine adenovirus,CAV),属于腺病毒科哺乳动物腺病毒属成员,是已发现的哺乳动物腺病毒属中致病性最强、形态结构研究得较清楚的一种动物病毒[1]。犬腺病毒广泛分布于全世界,是对我国养犬业、毛皮动物养殖业危害最大的疫病之一。CAV分为两种抗原相关型,即犬腺病毒I型(CAV-1)和犬腺病毒II型(CAV-2)。其中CAV-1称犬传染性肝炎病毒(CIHV),主要引起犬传染性肝炎和熊、狐脑炎。Loop1、Loop2为犬传染性肝炎病毒中和抗原决定簇,本研究设计并合成了Loop1和Loop2基因,连接两个基因命名为Loop ,①将Loop克隆到质粒载体pVAX1-CpG ,构建核酸疫苗pVAX1-CpG-Loop。采用脂质体体外转染,共聚焦观测结果显示构建的真核表达质粒可表达目的蛋白;采用ELISA法测定抗体效价,流式细胞检测CD4+和CD8+构成比,IFN-γ测定和中和抗体检测等实验方法,对pVAX1-CpG-Loop诱导小鼠的免疫效果进行了评价,结果显示各实验组小鼠均可产生体液免疫和细胞免疫应答。②将Loop基因片段克隆到质粒pET28a载体,构建原核表达质粒pET28a-Loop,表达Loop蛋白,制备了抗Loop蛋白的单克隆抗体,得到四株能产生中和抗体的克隆株,其中2-7F9,2-1B1中和抗体效价较高,可作为单抗诊断、治疗犬传染性肝炎的候选克隆株。
DNA vaccine is a new generation vaccine emerged in the early of 1990s and it has shown a great potential on the prevention and cure about a wide variety of diseases. DNA vaccine has some characteristics such as eliciting both cellular and humoral immune responses more completely than conventional vaccination and has the advantage of standardization easily. With the development of the promising technology, more and more nucleic acid vaccines, involving the prevention of animal infectious diseases, come into being. Because DNA vaccine seemed to be a promising novel approach for vaccination against canine infectious diseases, the DNA vaccine of pVAX1-CpG-Loop against hepatitis contagiosa canis virus was constructed and the immune efficiency was evaluated. Additionally, the monoclonal antibodies against the Loop of the virus were developed.
Hepatitis contagiosa canis was caused by hepatitis contagiosa canis virus, which is called Canine adenovirus (CAV) and belong to one number of adenovirus family. Hepatitis contagiosa canis virus have a powerful pathogenicity and its morphous and structure were studied thoroughly. Canine adenovirus (CAV) distributes worldwide and harm on canine and other fur animal cultivation. CAV was composed 2 different types, CAV-1 and CAV-2. CAV-1, which called ICHV, can cause hepatitis contagiosa canis and bear and fox encephalitis. Loop1 and Loop2 are epitopes of hepatitis contagiosa canis that can make virus neutralization. Loop1 and Loop2 were cloned respectively from hepatitis contagiosa canis virus, the fused gene Loop (Loop1-Loop2) was obtained and cloned into pVAX1-CpG vector. The protein expression of pVAX1-CpG-loop in B16 melanoma cells was detected by Western blot. The immune effiency of pVAX1-CpG-loop in mice was evaluated by ELISA and FACS. We found that vaccination with pVAX1-CpG-loop in mice resulted in both cellular and humoral immune responses were induced in overall mice and the IgG titer in combined immunization group was the highest. pET28a-Loop was constructed , Loop protein and monoclonal antibodies, 2-7F9,2-1B1,which were against Loop and can make virus neutralization, were obtained respectively. These results indicated that the availability of the DNA vaccine and antibodies can provide a technique platform for treatment of hepatitis contagiosa canis.
犬传染性肝炎是由犬传染性肝炎病毒感染引起。犬传染性肝炎病毒又称犬腺病毒(Canine adenovirus,CAV),属于腺病毒科哺乳动物腺病毒属成员,是已发现的哺乳动物腺病毒属中致病性最强、形态结构研究得较清楚的一种动物病毒[1]。犬腺病毒广泛分布于全世界,是对我国养犬业、毛皮动物养殖业危害最大的疫病之一。CAV分为两种抗原相关型,即犬腺病毒I型(CAV-1)和犬腺病毒II型(CAV-2)。其中CAV-1称犬传染性肝炎病毒(CIHV),主要引起犬传染性肝炎和熊、狐脑炎。Loop1、Loop2为犬传染性肝炎病毒中和抗原决定簇,本研究设计并合成了Loop1和Loop2基因,连接两个基因命名为Loop ,①将Loop克隆到质粒载体pVAX1-CpG ,构建核酸疫苗pVAX1-CpG-Loop。采用脂质体体外转染,共聚焦观测结果显示构建的真核表达质粒可表达目的蛋白;采用ELISA法测定抗体效价,流式细胞检测CD4+和CD8+构成比,IFN-γ测定和中和抗体检测等实验方法,对pVAX1-CpG-Loop诱导小鼠的免疫效果进行了评价,结果显示各实验组小鼠均可产生体液免疫和细胞免疫应答。②将Loop基因片段克隆到质粒pET28a载体,构建原核表达质粒pET28a-Loop,表达Loop蛋白,制备了抗Loop蛋白的单克隆抗体,得到四株能产生中和抗体的克隆株,其中2-7F9,2-1B1中和抗体效价较高,可作为单抗诊断、治疗犬传染性肝炎的候选克隆株。
DNA vaccine is a new generation vaccine emerged in the early of 1990s and it has shown a great potential on the prevention and cure about a wide variety of diseases. DNA vaccine has some characteristics such as eliciting both cellular and humoral immune responses more completely than conventional vaccination and has the advantage of standardization easily. With the development of the promising technology, more and more nucleic acid vaccines, involving the prevention of animal infectious diseases, come into being. Because DNA vaccine seemed to be a promising novel approach for vaccination against canine infectious diseases, the DNA vaccine of pVAX1-CpG-Loop against hepatitis contagiosa canis virus was constructed and the immune efficiency was evaluated. Additionally, the monoclonal antibodies against the Loop of the virus were developed.
Hepatitis contagiosa canis was caused by hepatitis contagiosa canis virus, which is called Canine adenovirus (CAV) and belong to one number of adenovirus family. Hepatitis contagiosa canis virus have a powerful pathogenicity and its morphous and structure were studied thoroughly. Canine adenovirus (CAV) distributes worldwide and harm on canine and other fur animal cultivation. CAV was composed 2 different types, CAV-1 and CAV-2. CAV-1, which called ICHV, can cause hepatitis contagiosa canis and bear and fox encephalitis. Loop1 and Loop2 are epitopes of hepatitis contagiosa canis that can make virus neutralization. Loop1 and Loop2 were cloned respectively from hepatitis contagiosa canis virus, the fused gene Loop (Loop1-Loop2) was obtained and cloned into pVAX1-CpG vector. The protein expression of pVAX1-CpG-loop in B16 melanoma cells was detected by Western blot. The immune effiency of pVAX1-CpG-loop in mice was evaluated by ELISA and FACS. We found that vaccination with pVAX1-CpG-loop in mice resulted in both cellular and humoral immune responses were induced in overall mice and the IgG titer in combined immunization group was the highest. pET28a-Loop was constructed , Loop protein and monoclonal antibodies, 2-7F9,2-1B1,which were against Loop and can make virus neutralization, were obtained respectively. These results indicated that the availability of the DNA vaccine and antibodies can provide a technique platform for treatment of hepatitis contagiosa canis.
引文
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