斑马鱼抗podocin抗体的制备、鉴定及在足细胞损伤模型中的应用  

Development and preliminary application of anti-zebrafish podocin antibody

作　　者：汪玲[1] 侯庆[1] 朱小东[1] 秦卫松[1] 曾彩虹[1] 陈朝红[1] 刘志红[1] 

机构地区：[1]南京大学医学院附属金陵医院(南京军区南京总医院)肾脏科国家肾脏疾病临床医学研究中心全军肾脏病研究所

出　　处：《肾脏病与透析肾移植杂志》2016年第2期146-152,共7页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：国家重点基础研究发展计划课题(2012CB517600;2012CB517606);国家自然科学基金(81470943);江苏省六大人才高峰(WSN-079-2013)

摘　　要：目的:利用合成的podocin多肽制备针对斑马鱼podocin的多克隆抗体,鉴定其特异性,并应用于斑马鱼足细胞损伤研究中。方法:(1)设计、合成斑马鱼podocin抗原多肽,将合成后的多肽与钥孔血蓝蛋白(KLH)偶联,免疫新西兰大白兔制备抗血清,protein A亲和纯化得到抗podocin多克隆抗体;(2)ELISA和免疫组化检测抗体效价及组织特异性;(3)构建足细胞特异性表达硝基还原酶(NTR)的转基因斑马鱼Tg(pod:Gal4;UAS:NTR-m Cherry),NTR/MTZ(甲硝唑)系统特异性地诱导足细胞损伤;用podocin反义寡核苷酸阻断正常斑马鱼胚胎podocin mRNA的翻译过程,利用抗podocin抗体对上述两种疾病模型进行免疫荧光染色以验证合成的podocin抗体能否应用于斑马鱼足细胞研究中。结果:(1)化学合成的多肽纯度为92.6%,达到免疫用抗原标准;(2)ELISA和免疫组化染色结果表明抗体效价分别达1∶5.12×105和1∶106;(3)免疫荧光染色表明无论在斑马鱼前肾还是中肾,podocin均特异性地表达于足细胞,且沿着毛细血管袢呈连续、线性分布,表明抗体具有良好的组织特异性;(4)MTZ诱导足细胞损伤24h后见podocin呈粗糙的颗粒状分布,48h后阳性表达面积占整个肾小球面积比值明显下降;(5)显微注射podocin反义寡核苷酸的斑马鱼胚胎出现心包水肿、体轴弯曲的表型,第3天时几乎无Podocin表达,随后逐渐恢复至正常,第5天时表达与正常组无异。结论:所制备的斑马鱼抗podocin抗体效价高、组织特异性强,能够反应足细胞损伤情况,是斑马鱼足细胞研究的有力工具。Objective： To prepare a polyclonal antibody against zebrafish podocin and evaluate podocyte injury with immunofluorescence using an in vivo zebrafish model of inducible podocyte injury. Methodology：（ 1） Zebrafish podocin peptides as antigen were designed and synthesized to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin（ KLH）. The polyclonal antibody in rabbit sera was purified by protein A column.（ 2） Titer and specificity of the polyclonal antibody was determined by ELISA and immunohistochemistry.（ 3） Transgenic zebrafish Tg（ pod; gal4; UAS： NTR-m Cherry） was generated as an inducible podocyte injury model in vivo by nitroreductase（ NTR） /metronidazole（ MTZ） system. Morpholino antisense oligonucleotides（ MO） was designed against translation of zebrafish embryo podocin mRNA. The synthesized podocin antibody was used to evaluate podocyte injury in the two podocyte injury models with immunofluorescence. Results：（ 1） The purify of synthesized podocin peptides was 92. 6%,which met the immunogen standard.（ 2） The ELISA and immunohistochemistry showed that the titers of anti-podocin antibody reached up to 1∶ 5. 12 × 105 and 1 ∶ 106.（ 3） The staining of podocin was revealed as fine contiguous linear-like pattern along the glomerular capillary loop both in normal pronephros and mesonephros of zebrafish.（ 4） After 24 hrs of MTZ treatment,the distribution of podocin became coarse granular pattern. The expression of podocin was significantly reduced at 48 h after MTZ treatment.（ 5） Podocin morpholino-injected embryos exhibited pericardial edema and body-axis curvature. The expression of podocin was remarkably decreased and only barely visible 3dpf. However,the expression and distribution of podocin was recovered gradually and completely restored to normal at 5dpf. Conclusion： The anti-zebrafish podocin antibody we prepared possesses high titer and specificity,and is correlated with the severity of podocyte injury,suggesting

关 键 词：斑马鱼 PODOCIN 足细胞 

分 类 号：R943[医药卫生—药剂学] R96[医药卫生—药学]