摘要
目的制备兔抗人STEAP4多克隆抗体及其特性鉴定。方法应用DNAStar软件对STEAP4的蛋白序列进行分析,选择STEAP4基因编码蛋白的近N端序列合成多肽并与牛血清白蛋白(BSA)交联。以纯化的STEAP4免疫新西兰白兔,制备抗STEAP4蛋白多抗,间接ELISA检测兔血清效价,Westernblot和免疫组织化学法鉴定多抗的特异性。结果得到兔抗人STEAP4多肽抗体,效价达16000。ELISA及蛋白印迹证实兔抗人STEAP4抗体可特异性识别STEAP4多肽。Westernblot结果显示该抗体识别的相应抗原的相对分子质量为52000。以一抗采用STEAP4多克隆抗体的脂肪组织石蜡切片为检测标本,进行STEAP4组织定位研究,发现STEAP4蛋白表达于脂肪细胞膜;以上结果皆与STEAP4的生物信息学分析一致。结论成功制备了抗STEAP4多克隆抗体,制备的兔抗人STEAP4合成肽抗体可应用于Westernblot和免疫组织化学试验,为后续深入研究STEAP4蛋白的功能奠定了基础。
Objective To prepare purify and characterize the polyclonal antibody against STEAP4. Methods The antigen epitopes of STEAP4 and its immunogenicity were analyzed and predicted by DNAStar software. A N - terminal partial peptide of STEAP4 was synthesized and coupled with bovine serum albumin （BSA）. The conjugate was used to immunize New Zealand rabbits. The sensitivity and specificity of polyclonal antibody against STEAP4 were conformed by ELISA,Western blot and immunohistoehemistry. Results The titer of the polyclonal antibody STEAP4 was 1 ： 6 000. Western blot confirmed its high specificity. It could be used for immunohistochemistry analysis. Western blot results showed that the antibody could recognize the protein with molecular weight of 52 000. Adipose tissue specimens of paraffin sections were used with the artibody for the detection. STEAP4 protein expression was found in the fat cell membrane. These results were consistent with analysis of bioinformatics. Conclusions The prepared polyclonal antibody against STEAP4 had been prepared,which could be used to Western blot and immunohistochemistry,which will be helpful to the further study of STEAP4.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2009年第19期1480-1483,共4页
Journal of Applied Clinical Pediatrics
基金
国家自然科学基金项目资助(30772364)
教育部基金项目资助(20070312001)
江苏省自然科学基金项目资助(BK2007230)
