血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达
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摘要
禽白血病(avian leukosis,AL)是指由反转录病毒科甲型反转录病毒属禽白血病病毒引起的以禽类造血组织中某些细胞成分过度增生为主的一类可传染的肿瘤性疾病,可划分为A-J 10个亚群。其中J亚群禽白血病病毒(ALV-J)是由内源性和外源性ALV重组而来,自发现以来给养禽业造成了巨大的经济损失。一直以来,由于ALV病毒群的多样性、病毒自身的特殊性以及引起疾病的复杂性,对ALV防治的研究未见显著成效,至今无控制ALV感染的有效方法、药物和疫苗。
J亚群白血病是由白血病病毒J亚群(ALV-J)引起的主要发生于肉种鸡的一种肿瘤性传染病,自1989年英国的Payne首次分离出ALV-J原型株HPRS-103以来,该病已迅速蔓延到世界各地,严重危害着养禽业的发展。近几年,我国数省26周龄左右的商品海兰褐蛋鸡爆发了血管瘤病,造成了巨大的经济损失。病鸡表现为衰竭、消瘦、在腿部、爪部、面部和翅下有大小不等的血疱。剖检可见内脏器官肿大,尤其是肝、脾表面及切面有大小不等的灰白色结节,内脏及体表血管瘤富有弹性,切开后流出大量的血液。
为了解血管瘤爆发的原因,寻找防制对策,本研究对诱发血管瘤的病毒进行分离鉴定,通过接种DF1细胞、特异性单抗的间接荧光抗体反应(IFA)实验,从患病鸡群中分离鉴定出J亚群禽白血病病毒(ALV-J),并将此病毒命名为WS0705。从而确证此次爆发的血管瘤与J亚群白血病病毒密切相关。为明确该型病毒特性,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示:该血管瘤毒株(WS0705)与国外4个ALV-J毒株同源性为84.4%～97.6%;与国内来源于白羽肉鸡的8株ALV-J病毒同源性在89.9%～94.5%;与本实验室分离的两株ALV-J毒株的同源性为96.6%～97.4%。其中与ALV-J原型株HPRS-103的同源性最高,达到97.6%。系统进化树表明该血管瘤型J亚型禽白血病病毒不同于中国其它ALV-J病毒,与英国ALV-J原型株HPRS-103亲缘关系最近。
为获得血管瘤型J亚群白血病毒gp85基因蛋白,为其快速精确诊断奠定基础,本研究利用昆虫杆状病毒表达系统对WS0705gp85基因进行表达。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacHTb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kDa的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中可以得到良好表达,且其编码产物完全可被外源性ALV-J的特异性单抗JE9识别。
Avian leukosis virus (ALV) is defined as alpha- retrovirus in poultry, it is a transmissible neoplastic viral groups which can induce hematopoietic cells hyperplasia. ALV can be divided into A-J 10 subgroups, in which subgroup J avian leukosis (ALV-J) is endogenous recombinate exogenous ALV, since the group has been found to cause significant economic losses.
Because of the diversity, the specificity of ALV virus and the lesion complexity caused by ALV, So far there is no the best way to prevent ALV, and no effective solutions,drugs and vaccines to control ALV infection.
Subgroup J avian leukosis (ALV-J) that is caused by avian leukosis virus subgroup J is a kind of tumors of horizontal and vertical transmission disease. This kind of disease mainly occurs in Broiler breeder chickens. Since 1989, Payne first time isolated ALV-J prototype strain HPRS-103 in the United Kingdom, the disease has spread rapidly around the world. In recent years, several outbreaks of hemangiomas in 26-week-old Hy-line Brown laying hens were observed in China. Hemangiomasthat induced by ALV-J caused great losses in egg-type chickens industry. The clinical manifestations were as follows: failure, weight loss, blood blisters in the legs, claws department, under the face and wings. In the necropsy, almost all viscera tissues showed moderate to severe enlargement with diffuse gray-white nodules; cutting open the hemangioma, blood effused and many myeloblasts were seen in blood smear.
For understanding the reasons for the outbreak of hemangioma, and finding control measures, the virus that induced hemangiomas was isolated and identified. By inoculating infected tissue suspension into DF1 cells and by indirect fluore scent assay ( IFA) with ALV-J specific monoclonal antibody JE-9,Subgroup J avian leukosis viruse (ALV-J ) was identified and isolated from sick chicken. The isolated strain was named ALV-J WS0705.
Gp85 gene was cloned into the pMD18-T vector and sequence was analyzed for better understand the antigenicity of the strain of ALV-J. Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain was the highest identification with the prototype HPRS-103(97.6%). Gp85 amino acid sequence comparison indicated that 84.4% ~ 97.6% identification with four international reference strains, 89.9%~94.5% indentification with eight Chinese strains from white meat-type chickens and 96.6% ~ 97.4% identification with two strains from ourlaboratory were identified. Phylogenetic tree showed that the gp85 amino sequence of WS0705 has the highest affinity with ALV-J prototype strain HPRS-103. The changes of pathotype induced by WS0705 in the layer chickens remained to be further studied.
WS0705 gp85 gene was expressed by baculovirus expression vector system. The protein expressed can be used to detect hemangiomas type ALV-J. The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector. rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system. The rBac-WS0705gp85 protein was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescent was present in Sf9 cells infected with recombinant virus and a protein band molecular weight about 35 kDa was present in western blot. It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with recombinant virus and the product WS0705 gp85 SU can bind specifically to JE9 MAb of ALV-J. The protein can be used to detect hemangiomas type ALV-J.
J亚群白血病是由白血病病毒J亚群(ALV-J)引起的主要发生于肉种鸡的一种肿瘤性传染病,自1989年英国的Payne首次分离出ALV-J原型株HPRS-103以来,该病已迅速蔓延到世界各地,严重危害着养禽业的发展。近几年,我国数省26周龄左右的商品海兰褐蛋鸡爆发了血管瘤病,造成了巨大的经济损失。病鸡表现为衰竭、消瘦、在腿部、爪部、面部和翅下有大小不等的血疱。剖检可见内脏器官肿大,尤其是肝、脾表面及切面有大小不等的灰白色结节,内脏及体表血管瘤富有弹性,切开后流出大量的血液。
为了解血管瘤爆发的原因,寻找防制对策,本研究对诱发血管瘤的病毒进行分离鉴定,通过接种DF1细胞、特异性单抗的间接荧光抗体反应(IFA)实验,从患病鸡群中分离鉴定出J亚群禽白血病病毒(ALV-J),并将此病毒命名为WS0705。从而确证此次爆发的血管瘤与J亚群白血病病毒密切相关。为明确该型病毒特性,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示:该血管瘤毒株(WS0705)与国外4个ALV-J毒株同源性为84.4%～97.6%;与国内来源于白羽肉鸡的8株ALV-J病毒同源性在89.9%～94.5%;与本实验室分离的两株ALV-J毒株的同源性为96.6%～97.4%。其中与ALV-J原型株HPRS-103的同源性最高,达到97.6%。系统进化树表明该血管瘤型J亚型禽白血病病毒不同于中国其它ALV-J病毒,与英国ALV-J原型株HPRS-103亲缘关系最近。
为获得血管瘤型J亚群白血病毒gp85基因蛋白,为其快速精确诊断奠定基础,本研究利用昆虫杆状病毒表达系统对WS0705gp85基因进行表达。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacHTb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kDa的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中可以得到良好表达,且其编码产物完全可被外源性ALV-J的特异性单抗JE9识别。
Avian leukosis virus (ALV) is defined as alpha- retrovirus in poultry, it is a transmissible neoplastic viral groups which can induce hematopoietic cells hyperplasia. ALV can be divided into A-J 10 subgroups, in which subgroup J avian leukosis (ALV-J) is endogenous recombinate exogenous ALV, since the group has been found to cause significant economic losses.
Because of the diversity, the specificity of ALV virus and the lesion complexity caused by ALV, So far there is no the best way to prevent ALV, and no effective solutions,drugs and vaccines to control ALV infection.
Subgroup J avian leukosis (ALV-J) that is caused by avian leukosis virus subgroup J is a kind of tumors of horizontal and vertical transmission disease. This kind of disease mainly occurs in Broiler breeder chickens. Since 1989, Payne first time isolated ALV-J prototype strain HPRS-103 in the United Kingdom, the disease has spread rapidly around the world. In recent years, several outbreaks of hemangiomas in 26-week-old Hy-line Brown laying hens were observed in China. Hemangiomasthat induced by ALV-J caused great losses in egg-type chickens industry. The clinical manifestations were as follows: failure, weight loss, blood blisters in the legs, claws department, under the face and wings. In the necropsy, almost all viscera tissues showed moderate to severe enlargement with diffuse gray-white nodules; cutting open the hemangioma, blood effused and many myeloblasts were seen in blood smear.
For understanding the reasons for the outbreak of hemangioma, and finding control measures, the virus that induced hemangiomas was isolated and identified. By inoculating infected tissue suspension into DF1 cells and by indirect fluore scent assay ( IFA) with ALV-J specific monoclonal antibody JE-9,Subgroup J avian leukosis viruse (ALV-J ) was identified and isolated from sick chicken. The isolated strain was named ALV-J WS0705.
Gp85 gene was cloned into the pMD18-T vector and sequence was analyzed for better understand the antigenicity of the strain of ALV-J. Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain was the highest identification with the prototype HPRS-103(97.6%). Gp85 amino acid sequence comparison indicated that 84.4% ~ 97.6% identification with four international reference strains, 89.9%~94.5% indentification with eight Chinese strains from white meat-type chickens and 96.6% ~ 97.4% identification with two strains from ourlaboratory were identified. Phylogenetic tree showed that the gp85 amino sequence of WS0705 has the highest affinity with ALV-J prototype strain HPRS-103. The changes of pathotype induced by WS0705 in the layer chickens remained to be further studied.
WS0705 gp85 gene was expressed by baculovirus expression vector system. The protein expressed can be used to detect hemangiomas type ALV-J. The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector. rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system. The rBac-WS0705gp85 protein was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescent was present in Sf9 cells infected with recombinant virus and a protein band molecular weight about 35 kDa was present in western blot. It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with recombinant virus and the product WS0705 gp85 SU can bind specifically to JE9 MAb of ALV-J. The protein can be used to detect hemangiomas type ALV-J.
引文
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