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摘要：GP85是ALV病毒表面的球状结构，能够识别靶细胞膜上的特异性受体，刺激机体产生中和抗体，是ALV亚群分类的主要依据和主要抗原蛋白。本论文主要探讨辽宁地方鸡-庄河大骨鸡的禽白血病病毒（ALV）的感染状况并对ALV-J gp85基因序列特点进行分析。本实验先采用ALV p27 ELISA试剂盒检测450份母鸡血清样品，从阳性样品鸡肝脏前病毒DNA进行ALV-J gp85基因的PCR扩增；将ALV-J阳性鸡肝脏滤过液接种DF-1细胞分离培养后，提取DNA进行gp85基因的PCR、克隆、测序，并与ALV-J原型株HPRS103和9个国内株序列进行比对。结果表明，辽宁地方鸡鸡群ALV检测阳性率为6.22%，ALV-J亚群仅占ALV的39.29%；ALV-J gp85 PCR测序长度为1622 bp，命名为LN17；LN17核苷酸序列与数据库10个分离株比较，发现与HPRS103相似度最高。本实验结果提示辽宁地方鸡鸡群存在ALV感染，不仅有ALV-J亚群，还有其它类型；ALV-J gp85 LN17与原型株HPRS103相似程度最高。28050
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毕业论文关键词：辽宁地方鸡；ALV；gp85；核苷酸相似性比较
Sequence variation of gp85 gene of avian leukosis virus subgroup J in Liaoning native chicken
Abstract: GP85 was a globular structure on the surface of avian influenza viruses (ALV), which identified the specific receptor on the target cells and stimulated the production of neutralizing antibody. GP85 was the standard of the subgroup classification and the main antigen protein. This thesis mainly investigated the infection level of ALV and learned the sequence characteristics of ALV-J gp85 gene in Liaoning native chicken-Zhuanghe Dagu chicken. In this study, ALV p27 ELISA kits were used to detect infection of ALV in 450 hens serum. Liver virus pre-DNA of ELISA positive samples were extracted and ALV-J gp85 gene was amplified by PCR. Liver filtrate of ALV-J sample was cultured in DF-1 cells and virus pre-DNA was extracted for PCR, cloning and sequencing of gp85 gene. The nucleotide sequence we acquired was compared with typical strain HPRS103 and 9 domestic strains. As a result, Liaoning local hens were infected with ALV and the positive rate was 6.22%. Subgroup J of ALV was accounted 39.29% in positive samples. Length of ALV-J gp85 by sequencing which named LN17 was 1622bp. Similarity nucleotide comparison between LN17 and HPRS103 and 9 isolated domestic strains showed that LN17 had the highest score with HPRS103. In the conclusion, there was ALV infection in Liaoning local chicken flocks with ALV-J and other subgroups. LN17 strain of ALV-J gp85 had the highest degree of similarity with the prototype strain HPRS103.
Key words: Liaoning native chicken; ALV-J; gp85; Nucleotide similarity comparison
目 录
摘要1
关键词1
Abstract1
Key words2
引言（或绪论）2
1 材料与方法2
1.1 实验材料 2
1.1.1 实验鸡群和样品采集2
1.1.2 主要试剂2
1.1.3 主要实验仪器3
1.1.4 主要分子生物学和统计学软件3
1.2 实验方法 3
1.2.1 血清ALV p27抗原ELISA检测3
1.2.2 鸡肝脏ALV-J前病毒DNA提取和gp85 PCR扩增和电泳3
1.2.3 病毒的体外分离培养及培养物中前病毒DNA的提取3
1.2.4 gp85基因的扩增、产物回收纯化3
1.2.5 PCR产物的T-A克隆和测序4
1.2.6 ALV-J亚群gp85基因序列分析4
2 结果与分析4
2.1 试验鸡群及初产蛋5
2.2 血清ALV p27抗原ELISA检测5
2.3 阳性母鸡肝脏前病毒DNA的ALV-J gp85 PCR扩增和电泳6
2.4 ALV-J 的细胞分离培养8
2.5 ALV-J亚群gp85基因PCR扩增产物的T-A克隆和测序9
2.6 ALV-J分离株LN17与参考株的同源性和遗传进化树分析10
3 讨论11
3.1 ALV p27 ELISA的检测方法11
3.2 ALV-J亚群gp85的基因变异12