摘要
目的:制备鸡类固醇调节元件结合蛋白1(SREBP1)的多克隆抗血清,并分析其组织表达特性。方法:利用DNAStar软件对鸡SREBP1入核部分蛋白进行分析,预测其主要抗原决定簇区域;利用RT-PCR的方法扩增编码鸡SREBP1主要抗原决定簇的基因片段(832-1 302 bp),构建原核表达载体pGEX-4T/SREBP1;诱导重组蛋白GST/SREBP1表达并纯化后,以其为免疫原制备鸡SREBP1的多克隆抗血清;通过ELISA和Western blot的方法检测抗血清的效价和特异性,并利用此抗血清分析SREBP1基因在肉鸡组织中的表达特性。结果:ELISA测定抗体效价为1∶102 400,Western blot结果显示抗体具有较高的特异性;SREBP1基因在肉鸡腹部脂肪组织和心脏组织中有较高表达,在肝脏、肌胃、十二指肠、脾脏、肾脏等组织表达量较低,在胸肌和腿肌中没有检测到信号;SREBP1基因在高脂系公鸡腹部脂肪组织中的表达量显著高于低脂系公鸡腹部脂肪组织的表达量(P<0.05)。结论:本研究制备的鸡SREBP1的多克隆抗血清效价高、特异性强。SREBP1在肉鸡腹部脂肪组织高量表达。与低脂系公鸡相比,SREBP1在高脂系公鸡腹部脂肪组织的表达量较高。
AIM： The aim of this study was to prepare the antiserums against chicken sterol regulatory element binding protein1（SREBP1）,and to analyze the expression of SREBP1 in chicken tissues.METHODS： The nuclear import sequence of SREBP1 was analyzed using the DNAStar programs to predict its major antigen epitopes,the fragment coding for SREBP1 major antigen epitopes（832-1 302 bp） was amplified by RT-PCR and inserted into pGEX-4T-1 to construct the expression vector pGEX-4T/SREBP1.The recombinant GST/SREBP1 was expressed in E.coli with IPTG induction and purified by Glutathione Sepharose 4B affinity chromatography.The purified recombinant GST/SREBP1 was used as immunogen in rabbits,and the titer and specificity of SREBP1 antiserums were detected by ELISA and Western blot.The tissue expression of chicken SREBP1 was analyzed with the antiserums.RESULTS： The titer of the antiserum determined by ELISA was 1∶102 400,and the antiserums exhibited a high specificity through Western blot.Tissue expression analysis showed that the expression of chicken SREBP1 was much higher in abdominal adipose tissue and heart,but lower in liver,muscle stomach,duodenum,spleen,kidney,and no signal was detected in breast and leg muscle.The SREBP1 expression characteristics between fat and lean broiler lines was also detected,and the result showed that SREBP1 expressed significantly higher in the abdominal adipose tissue of fat line than that in lean line（P0.05）.CONCLUSION： The antiserum against chicken SREBP1 prepared in this study had high titer and specificity.SREBP1 was highly expressed in abdominal adipose tissue of broilers.Compared to lean broiler line,SREBP1 was expressed higher in the fat line.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第12期1241-1245,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展计划(973)资助项目(2009CB941604)
国家高技术研究发展计划(863)资助项目(2006AA10A120)
黑龙江省自然科学基金重点项目(ZJN0604-01)
关键词
鸡
SREBP1
抗血清
表达
特性
Chicken
SREBP1
antiserum
expression
characteristics
