鹅热休克蛋白70的表达提纯与病毒复合物形成的研究  

Expression and Purification of Goose's HSP70 and Complex Formation with Virus Polypeptides

作　　者：吴晓平[1] 王宝维[1] 张旭晖[1] 王斌[1] 贾晓晖[1] 张名爱[1] 龙芳羽[1] 杨志刚[1] 王雷[1] 

机构地区：[1]青岛农业大学优质水禽研究所,青岛266109

出　　处：《中国农业科学》2007年第9期2084-2090,共7页Scientia Agricultura Sinica

基　　金：国家"十五"科技攻关项目(2002BA514A20)

摘　　要：【目的】为研究HSP70在热应激时的特异性表达、组织中HSP70的分离纯化及其结合抗原肽形成复合物的特性,设计此试验。【方法】选取6w健康五龙鹅60只随机分为3组,对照组鹅不经热处理,试验1组鹅进行1次(42±1)℃、5h的急性热处理后立即宰杀,试验2组鹅经热处理后在正常饲养条件下恢复12h后宰杀。取心脏,常规石蜡切片做免疫组化试验;肝脏用于提纯HSP70。比较处理组鹅心脏HSP70表达的差异,同时对肝脏中HSP70进行分离纯化、鉴定。纯化后的HSP70和人工合成的乙肝病毒前S1多肽相结合形成复合物,以双特异抗体夹心法检测其复合物的形成。【结果】热应激状态下心肌纤维中有广泛的HSP70阳性颗粒,试验1组HSP70表达部位重点在核膜及其周围,阳性细胞率为76.0%,试验2组HSP70表达部位转移到胞膜周围,阳性细胞率为88.4%。通过两套纯化方案纯化蛋白均可得到HSP70纯化物,蛋白纯度分别为85.0%和95.0%,蛋白得率分别为0.115mg·g-1和0.157mg·g-1。一定条件下合成肽与HSP70纯化物形成复合物,双特异性抗体夹心法检测复合物为阳性,复合物效价为1﹕81,表明试验中有复合物的形成。【结论】热应激时HSP70的特异性表达部位,在不同时间有所转移,显示HSP70在细胞保护中可能具有一定的功能。在低压层析条件下可以得到高纯度的HSP70蛋白,相比较而言,分子筛初步分离、ConA琼脂糖层析和ADP琼脂糖层析这一流程的纯化效果更好。在一定条件下,人工合成肽可以与HSP70相结合形成复合物,这将为进一步证明复合物的免疫作用提供试验基础。　【Objective】 In order to study the specificity expression of HSP70 in heat shock,HSP70 purification and its characteristics of coalescent with antigen peptide to form the compound,we designed this experiment.【Method】 Sixty healthy,6-week-old male Wulong geese were selected and randomly divided into 3 groups.The geese in control group were not treated by heat.Group 1 was shocked by 1 time（42±1）℃,5h acute heat treatment and then were slaughtered immediately.Group 2 was restored 12h after the heat treatment under the normal raising conditions and then were slaughtered.Hearts were taken to make paraffin section for the immunization organizations chemical experiment and livers were used to purify HSP70.The difference in geese hearts HSP70 expression in 3 groups was determined,at the same time experiments of HSP70 purification and appraisal in the liver was carried out.The purified HSP70 and synthetic HBV PreS1 multi-peptides formed the compound,and was determined by the double special immune body ELISA.【Result】 The findings indicated that,widespread HSP70 masculine pellets in the cardic muscle were found at hot shock conditions,HSP70 expression in treatment group 1 centered on the karyotheca and its periphery,but treatment group 2 centered on the cell membrane around;The HSP70 purification was obtaind through two sets of purification plans;the synthesis peptide and the HSP70 purification forms the compound under certain conditions,examination taken to compound formation with the double specificity immune body,and the examination result was positive,which explained that the compound had formed.【Conclusion】 HSP70 specificity expression in heat shock shifted by time,and it may suggest that HSP70 possibly do some certain functions in the cell protection.High-purity HSP70 protein can be obtained under low pressure chromatographic analysis condition.In comparission,it is better than the molecular sieve preliminary separation,ConA-agarose chromatographic analysis and the ADP-agarose chromatographical

关 键 词：热应激 鹅 HSP70 表达 提纯 病毒复合物 

分 类 号：S852.2[农业科学—基础兽医学]