用硫酸铵分级盐析法纯化大菱鲆血清免疫球蛋白IgM,所得产物用Sepharose-4B和DEAE-Sepharose Fast Flow阴离子交换层析进一步纯化,以纯化的大菱鲆IgM免疫新西兰大白兔,获得兔抗大菱鲆IgM抗血清。SDS-PAGE电泳显示大菱鲆IgM重链为76 kD,...用硫酸铵分级盐析法纯化大菱鲆血清免疫球蛋白IgM,所得产物用Sepharose-4B和DEAE-Sepharose Fast Flow阴离子交换层析进一步纯化,以纯化的大菱鲆IgM免疫新西兰大白兔,获得兔抗大菱鲆IgM抗血清。SDS-PAGE电泳显示大菱鲆IgM重链为76 kD,轻链为27 kD;纯化的大菱鲆IgM重链与特异性抗血清具有较好的反应,而轻链与抗血清反应不明显;以制备的兔抗大菱鲆IgM抗血清为二抗建立了大菱鲆血清特异性抗体的间接ELISA检测方法,用该方法检测了鳗弧菌灭活疫苗免疫后大菱鲆产生特异性抗的变化规律,大菱鲆在免疫后第1周就产生了特异性抗体,在3周时达到峰值,该特异性抗体可维持13周以上。展开更多
采用盐析法、重组蛋白A(HiTrapr Protein A Sepharose)亲和层析法分离纯化草鱼血清中的IgM,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较并制备兔抗IgM抗血清。结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,...采用盐析法、重组蛋白A(HiTrapr Protein A Sepharose)亲和层析法分离纯化草鱼血清中的IgM,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较并制备兔抗IgM抗血清。结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,但电泳条带仍较多,其中含有78kD和28kD的条带,因此仅可作为免疫球蛋白粗提的方法;而rProtein A亲和层析法所提蛋白则仅有上述重链(78kD)和轻链(28kD)。Western-blot显示,鼠抗人Ig抗体可与78kD及28kD条带发生发应。rProtein A亲和法提纯蛋白的纯度较高,但含量较低,条带较淡,仅可作为实验室小量提纯草鱼IgM的有效方法。将提纯的蛋白免疫实验兔后可制得效价高达1:25600的兔抗鱼IgM血清,并测得血清蛋白总量和IgM含量分别为25.87mg和4.5mg,IgM占血清蛋白总量的17.39%。本实验所采用的蛋白A亲和层析法提取草鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类IgM。同时本研究所制备的兔抗草鱼IgM血清也为今后的相关研究工作打下基础。展开更多
Central Composite Design(CCD) and response surface methodology were used in the experiment to examine the combined effect of temperature(16-28℃) and salinity(18-42) on Hsp70 and IgM genes expression levels in turbot(...Central Composite Design(CCD) and response surface methodology were used in the experiment to examine the combined effect of temperature(16-28℃) and salinity(18-42) on Hsp70 and IgM genes expression levels in turbot(Scophthalmus maximus) liver and kidney.The results showed that the coefficients of determination(R^2=0.965 2 for liver Hsp70,0.972 9 for kidney Hsp70,0.921 for liver IgM and 0.962 1 for kidney IgM) and probability values(P<0.01) were significant for the regression model.The interactive effect between temperature and salinity on liver Hsp70,kidney Hsp70 and liver IgM were not significant(P>0.05),while the interactive effect between temperature and salinity on kidney IgM was significant(P<0.01).The model equation could be used in practice for forecasting Hsp70 and IgM genes expression levels in the liver and kidney of juvenile turbot via applying statistical optimization of the response of interest,at which the maximum liver Hsp70,kidney Hsp70,liver IgM and kidney IgM of1.48,1.49,2.48,and 1.38,respectively,were reached.The present model may be valuable in assessing the feasibility of turbot farming at different geographic locations and,furthermore,could be a useful reference for scientists studying the immunity of turbot.展开更多
文摘用硫酸铵分级盐析法纯化大菱鲆血清免疫球蛋白IgM,所得产物用Sepharose-4B和DEAE-Sepharose Fast Flow阴离子交换层析进一步纯化,以纯化的大菱鲆IgM免疫新西兰大白兔,获得兔抗大菱鲆IgM抗血清。SDS-PAGE电泳显示大菱鲆IgM重链为76 kD,轻链为27 kD;纯化的大菱鲆IgM重链与特异性抗血清具有较好的反应,而轻链与抗血清反应不明显;以制备的兔抗大菱鲆IgM抗血清为二抗建立了大菱鲆血清特异性抗体的间接ELISA检测方法,用该方法检测了鳗弧菌灭活疫苗免疫后大菱鲆产生特异性抗的变化规律,大菱鲆在免疫后第1周就产生了特异性抗体,在3周时达到峰值,该特异性抗体可维持13周以上。
文摘采用盐析法、重组蛋白A(HiTrapr Protein A Sepharose)亲和层析法分离纯化草鱼血清中的IgM,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较并制备兔抗IgM抗血清。结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,但电泳条带仍较多,其中含有78kD和28kD的条带,因此仅可作为免疫球蛋白粗提的方法;而rProtein A亲和层析法所提蛋白则仅有上述重链(78kD)和轻链(28kD)。Western-blot显示,鼠抗人Ig抗体可与78kD及28kD条带发生发应。rProtein A亲和法提纯蛋白的纯度较高,但含量较低,条带较淡,仅可作为实验室小量提纯草鱼IgM的有效方法。将提纯的蛋白免疫实验兔后可制得效价高达1:25600的兔抗鱼IgM血清,并测得血清蛋白总量和IgM含量分别为25.87mg和4.5mg,IgM占血清蛋白总量的17.39%。本实验所采用的蛋白A亲和层析法提取草鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类IgM。同时本研究所制备的兔抗草鱼IgM血清也为今后的相关研究工作打下基础。
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2012AA10A408-8)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.CARS-50-G01)
文摘Central Composite Design(CCD) and response surface methodology were used in the experiment to examine the combined effect of temperature(16-28℃) and salinity(18-42) on Hsp70 and IgM genes expression levels in turbot(Scophthalmus maximus) liver and kidney.The results showed that the coefficients of determination(R^2=0.965 2 for liver Hsp70,0.972 9 for kidney Hsp70,0.921 for liver IgM and 0.962 1 for kidney IgM) and probability values(P<0.01) were significant for the regression model.The interactive effect between temperature and salinity on liver Hsp70,kidney Hsp70 and liver IgM were not significant(P>0.05),while the interactive effect between temperature and salinity on kidney IgM was significant(P<0.01).The model equation could be used in practice for forecasting Hsp70 and IgM genes expression levels in the liver and kidney of juvenile turbot via applying statistical optimization of the response of interest,at which the maximum liver Hsp70,kidney Hsp70,liver IgM and kidney IgM of1.48,1.49,2.48,and 1.38,respectively,were reached.The present model may be valuable in assessing the feasibility of turbot farming at different geographic locations and,furthermore,could be a useful reference for scientists studying the immunity of turbot.
