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(附件)【字数：8068】
3
目 录
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
1.3BS10VP2 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鸡胚成纤维细胞(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vHVT05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vHVT05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
动物医学专业学生 郭淑敏
指导教师 严若峰
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UL44UL45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pUABgpt。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pUABgpt，获得转移载体质粒pUABPecBS10VP2gpt。将该HVT转移载体与HVT DNA共转染鸡胚成纤维细胞(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rHVTPecBS10VP2。Western blotting分 *好棒文|www.hbsrm.com +Q: ￥351916072$
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。
Construction of recombinant Turkey Herpesvirus expressing infectious bursal disease virus VP2 and bursal cysteoid chimeric gene
Student majoring in Veterinary medicine Shumin Guo
Tutor Ruofeng Yan
Abstract: The construction of recombinant turkey herpesviruses of infectious bursal disease virus VP2 and bovine cysteine tripeptide chimeric gene was studied. Non essential region UL44UL45 of HVT FC126 genome was selected as insertion sites. Using the selection marker Ecogpt Xanthineguanine phosphoribosyl transferase (gpt), plasmid pUABgpt were constructed. Then, BS10VP2 expression cassette was constructed containing synthesised Pec promoters and then was cloned into the plasmid pUABgpt to obtain the transfer vector of turkey herpesvirus pUABPecBS10vp2gpt. 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 rHVTPecBS10VP2 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.
目 录
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
1.3BS10VP2 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鸡胚成纤维细胞(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vHVT05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vHVT05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
动物医学专业学生 郭淑敏
指导教师 严若峰
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UL44UL45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pUABgpt。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pUABgpt，获得转移载体质粒pUABPecBS10VP2gpt。将该HVT转移载体与HVT DNA共转染鸡胚成纤维细胞(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rHVTPecBS10VP2。Western blotting分 *好棒文|www.hbsrm.com +Q: ￥351916072$
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。
Construction of recombinant Turkey Herpesvirus expressing infectious bursal disease virus VP2 and bursal cysteoid chimeric gene
Student majoring in Veterinary medicine Shumin Guo
Tutor Ruofeng Yan
Abstract: The construction of recombinant turkey herpesviruses of infectious bursal disease virus VP2 and bovine cysteine tripeptide chimeric gene was studied. Non essential region UL44UL45 of HVT FC126 genome was selected as insertion sites. Using the selection marker Ecogpt Xanthineguanine phosphoribosyl transferase (gpt), plasmid pUABgpt were constructed. Then, BS10VP2 expression cassette was constructed containing synthesised Pec promoters and then was cloned into the plasmid pUABgpt to obtain the transfer vector of turkey herpesvirus pUABPecBS10vp2gpt. 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 rHVTPecBS10VP2 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.
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