表达传染性法氏囊病毒VP2和法氏囊素三肽嵌合基因重组火鸡疱疹病毒的构建

摘要：本课题研究了传染性法氏囊病（IBD）重组火鸡疱疹病毒的构建策略。以HVT FC126基因组的非必需区UL44-UL45为插入位点，构建带有大肠杆菌黄嘌吟-鸟嘌吟磷酸转移酶基因序列(gpt)标记的有HVT转移载体质粒pUAB-gpt。然后由人工合成Pec启动子，构建表达传染性法氏囊病毒（IBDV）VP2嵌合法氏囊素三肽（BS10）表达盒并克隆入pUAB-gpt，获得转移载体质粒pUAB-Pec-BS10-VP2-gpt。将该HVT转移载体与HVT DNA共转染鸡胚成纤文细胞(CEF)，待出现病毒噬斑后，利用霉酚酸(MPA)阻断核酸代谢途径，经过筛选获得重组病毒rHVT-Pec-BS10-VP2。Western blotting分析，在53 kDa处出现一条特异蛋白条带。IBDV抗体夹心ELISA检测，重组病毒呈阳性反应，抗原效价达1.6×103。结果表明，IBD重组火鸡疱疹病毒构建成功。28051
毕业论文关键词：鸡传染性法氏囊病；VP2基因；法氏囊素三肽；重组火鸡疱疹病毒
Construction of recombinant Turkey Herpesvirus expressing infectious bursal disease virus VP2 and bursal cysteoid chimeric gene
Abstract: The construction of recombinant turkey herpesviruses of infectious bursal disease virus VP2 and bovine cysteine tripeptide chimeric gene was studied. Non essential region UL44-UL45 of HVT FC126 genome was selected as insertion sites. Using the selection marker Eco-gpt Xanthine-guanine phosphoribosyl transferase (gpt), plasmid pUAB-gpt were constructed. Then, BS10-VP2 expression cassette was constructed containing synthesised Pec promoters and then was cloned into the plasmid pUAB-gpt to obtain the transfer vector of turkey herpesvirus pUAB-Pec-BS10-vp2-gpt. Finall, transfer vector and total DNA of HVT infected cells were cotransfected into chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, recombinant virus rHVT-Pec-BS10-VP2 was obtained. Western blotting revealed that the calculated protein of approximately 53 kDa was expressed in the CEF cells. and recombinant virus was positive with antigen titer of 1.6×103 by IBDV sandwich ELISA. Recombinant IBD turkey herpesvirus is proved successful.
Key words: IBDV; VP2gene ;BS10; recombinant rHVT
目录

摘要3
关键词3
Abstract3
Key words3
引言4
1材料与方法4
1.1主要试剂 5
1.2 SPF鸡胚和病毒5
1.3BS10-VP2 基因设计、克隆及表达盒的构建5
1.4重组火鸡疱疹病毒转移载体的构建5
1.5重组火鸡疱疹病毒的的获得6
1.5.1鸡胚成纤文细胞(CEF)的制备6
1.5.2脂质体转染6
1.5.3重组火鸡疱疹病毒的筛选6
1.6重组病毒vHVT-05株的生物学特性7
1.7夹心ELISA检测7
1.8 Western blotting分析7
2结果与分析7
2.1重组火鸡疱疹病毒转移载的构建7
2.2重组火鸡疱疹病毒病毒的获得8
2.3重组火鸡疱疹病毒的鉴定9
2.3.1夹心ELISA检测9
2.3.2Western blotting分析9
2.4重组病毒与野生病毒在CEF上的生长曲线比较10
2.5重组病毒vHVT-05株的遗传稳定性10
3讨论 11
致谢12
参考文献13
表达传染性法氏囊病毒VP2和法氏囊素三肽嵌合基因重组火鸡疱疹病毒的构建
引言
传染性法氏囊病(Infectious bursal disease，IBD) 是鸡、鸭、鹅等家禽中最常见的一种疾病之一，它能影响家禽的生长繁殖，从而影响家禽的生产，对家禽养殖业的发展造成极大的危害。传染性法氏囊病IBD是一种免疫抑制性疾病，是由传染性法氏囊病病毒(Infectious bursal disease virus，IBDV)引起的鸡免疫抑制性传染病，它的主要特点就是传染非常迅速，并且呈大面积传染，爆发非常快，在群体饲养的鸡当中传播超级快。IBD的免疫抑制产生时，常常会伴有有新城疫（ND）、马立克氏病（MD）等疫苗接种的免疫效果降低，更甚至会导致他们的免疫失败，这种免疫抑制导致这些疾病的发病率和病死率升高，给养鸡业造成非常大危害[1-2]。表达传染性法氏囊病毒VP2和法氏囊素三肽嵌合基因重组火鸡疱疹病毒的构建:http://www.751com.cn/yixue/lunwen_22760.html