摘要
目的 制备特异性识别人谷氨酰胺果糖6磷酸氨基转移酶1(GFPT1)的兔多克隆抗体。方法 通过比对GFPT1及其高度同源的同工酶GFPT2的蛋白质序列,针对GFPT1的特异性序列设计并合成2个多肽,多肽与钥孔戚血蓝蛋白(KLH)偶联作为抗原免疫新西兰大白兔,3次加强免疫后得到抗血清,采用ELISA检测抗血清效价。构建GFPT1和GFPT2的大肠杆菌表达载体,诱导表达获得GFPT1和GFPT2重组蛋白,通过Western blot法检测GFPT1和GFPT2重组蛋白以验证抗血清的特异性、免疫荧光细胞化学染色检测786-O细胞GFPT1的表达验证获得的抗血清能否识别内源性表达的GFPT1。结果 获得了特异性识别GFPT1的多克隆抗体,效价高达1∶1 458 000,该抗体可用于Western blot法以及免疫荧光细胞化学染色。结论 成功制备了特异性识别GFPT1的兔多克隆抗体。
Objective To prepare rabbit polyclonal antibody specifically against human glutamine fructose-6-phosphate amidotransferase 1(GFPT1). Methods The protein sequences of GFPT1 and its highly homologous isozyme GFPT2 were compared. Two peptides for the specific sequence of GFPT1 were designed and synthesized. New Zealand rabbits were immunized by peptide coupled with Keyhole Limpet hemocyanin(KLH) as antigen. Antiserum was obtained after 3 booster immunizations. The titer of the antiserum against GFPT1 were detected by ELISA. The E.coli expression vectors of GFPT1 and GFPT2 were constructed, and the recombinant proteins of GFPT1 and GFPT2 were obtained by induced expression. GFPT1 and GFPT2 recombinant proteins were analyzed by Western blot to verify the specificity of the antiserum. Immuno-fluorescence cytochemical staining for GFPT1 expression in 786-O cells was verified as for whether the obtained antiserum could recognize the endogenously expressed GFPT1. Results Polyclonal antibody specifically recognizing GFPT1 was obtained and the titer of polyclonal antibody reached 1∶1 458 000. Conclusion The experiment successfully prepared the specific rabbit polyclonal antibody against GFPT1.
作者
吴少春
刘林
高艳春
顾玉超
WU Shaochun;LIU Lin;GAO Yanchun;GU Yuchao(School of Medicine and Pharmacy,Ocean University of China,Qingdao 266000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第7期651-656,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81871868)
中央高校基本科研业务费(202042011)。
