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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。
Objective: To prepare polyclonal antiserum of chicken steroid regulatory element binding protein 1 (SREBP1) and to analyze its tissue expression characteristics. Methods: The DNA of SREBP1 was analyzed by DNAStar software to predict its main epitopes. The gene fragment (832-1 302 bp) coding for the major antigenic determinant of chicken SREBP1 was amplified by RT-PCR and constructed The prokaryotic expression vector pGEX-4T / SREBP1 was constructed. After the recombinant protein GST / SREBP1 was expressed and purified, it was used as an immunogen to prepare polyclonal antiserum against chicken SREBP1. The antiserum titer and specificity were determined by ELISA and Western blot , And using this antiserum to analyze the expression characteristics of SREBP1 gene in broiler tissues. Results: The titer of the antibody was 1:102 400 by ELISA. The result of Western blot showed that the antibody was highly specific. The SREBP1 gene was highly expressed in adipose tissue and heart tissues of broilers, The expression of SREBP1 gene in abdomen adipose tissue of high-fat rooster group was significantly higher than that of low-fat rooster belly fat tissue (P <0.05). Conclusion: The polyclonal antiserum of chicken SREBP1 prepared in this study has high potency and specificity. SREBP1 is highly expressed in the abdominal adipose tissue of broilers. SREBP1 expressed higher levels of adipose tissue in the abdomen of high-fat rooster than low-fat rooster.