摘要
目的:利用原核表达系统重组表达小鼠附睾特异性辅脂酶(meClps),并制备抗meClps抗体。方法:通过生物信息分析meClps蛋白结构,Oligo 6设计引物,PCR法从小鼠附睾cDNA中扩增meClps全长序列并克隆到pMD19-T载体中。将含meClps序列pMD19-T载体通过PCR扩增成熟肽区段序列,重组到pET-21b原核表达载体上,经菌落PCR及测序鉴定后,转化到表达菌株E.coliBL21(DE3),经IPTG诱导蛋白表达后用Tricine-SDS-PAGE和Western blotting检测重组meClps的表达。包涵体沉淀经浓度为2 mol/L尿素洗涤,Tricine-SDS-PAGE分离、染色、脱色,切下目的条带用生理盐水浸泡、磨碎与弗氏佐剂混合后免疫新西兰大白兔获取免疫血清,Western blotting检测抗体与meClps反应。结果:在原核表达系统成功重组表达meClps,目的蛋白主要以包涵体形式存在,制备了高效价的兔抗meClps抗体。结论:成功建立meClps的原核表达系统和获得兔抗meClps抗体。
Aim：To express mouse epididymis-specific colipase（meClps） in prokaryotic system and prepare the anti-meClps antibody. Methods： The meClps protein structure was analysed by using bioinformation,and primers were designed with Oligo 6.The full-length of meClps was amplified from mouse epididymal cDNA and cloned into pMD19-T vector.The sequence of mature peptide was obtained by PCR from pMD19-T vector with meClps.Then the product was recombined into the prokaryotic expression vector pET-21b.The recombinant plasmid was identified by clone PCR and sequencing before transformation into E.coli BL21（DE3）.After induced by IPTG,the recombinant meClps protein was expressed and sepreated by Tricine-SDS-PAGE,and then identified by Western blotting.The inclusion bodies were washed with 2 mol/L urea and sepreated by Tricine-SDS-PAGE.After staining and destaining,the gel containing meClps protein was cut,equilibrated with physiological saline solution,rubbed,and emulsified with Freundadjuvant,then used as antigen to immunize rabbit and prepare antibodies.The anti-meClps antibody response was identified by Western blotting.Results： The meClps protein was successfully expressed in prokaryotic system,but mainly in inclusion bodies.The rabbit anti-meClps antibody was prepared with high potency.Conclusion：The prokaryotic expression system for the meClps protein was established and rabbit anti-meClps antibody was obtained,which provides the possibility for functional study of meClps.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第2期105-110,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金项目(30570683)
关键词
附睾
辅脂酶
原核表达
抗体
epididymis
colipase
prokaryotic expression
antibody
