摘要
目的:表达并纯化小鼠釉成熟蛋白(Amelotin),制备多克隆抗体,并进行初步鉴定。方法:RT-PCR获得Amelo-tin成熟肽片段,构建pET32a-Amelotin,IPTG诱导表达Amelo-tin,纯化后免疫新西兰大白兔制备多克隆抗体,ELISA检测抗体滴度。Western blot和免疫组织化学鉴定抗体特异性。结果:成功构建了Amelotin重组表达载体pET32a-Amelotin,表达的重组蛋白纯化后免疫新西兰大白兔,得到的多克隆抗体ELISA显示抗体效价可以达到1∶12800。Western blot分析表明该抗体能特异结合Amelotin,免疫组化分析表明自制的抗体能特异的与Amelotin相互作用。结论:以纯化的Amelo-tin为免疫原,成功地制备了效价及特异性较高的兔抗Amelo-tin抗体,为进一步建立简便的Amelotin检测方法及研究Amelotin生物学功能奠定了良好的基础。
AIM： To construct a mouse Amelotin recombinant plasmid, express mouse Amelotin protein and prepare its polyclonal antibody. METHODS： The cDNA sequence of mouse Amelotin gene was downloaded from Gen-Bank. The coding region, which did not include signal peptide, was amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a and transformed into E. coli BL21 （DE3）. After induced by IPTG, the recombinant protein was expressed and purified using affinity purification. The polyclonal antibody was obtained from New Zealand rabbit immunized with the recombinant protein and the titer was identified by ELISA. The polyclonal antibody was identified by Western blot and immunohistochemistry assays. RESULTS： The recombinant prokaryotic expression vector pET32a-Amelotin was constructed and the recombinant prorein was purified successfully. ELISA analysis showed the titer of the generated antiserum was 1：12 800. Western blot and immunohistochemistry analysis demonstrated this antibody bound specifically with Amelotin. CONCLUSION： The anti-Amelotin antibody from the rabbit with high titer and specificity had been prepared with the purified recombinant Amelotin as immunogen, which is helpful for further research into the detection and function of Amelotin.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第4期328-331,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30672316)
山东省教育厅重点项目(J06L22)
关键词
釉成熟蛋白
原核表达
多克隆抗体
Amelotin
prokaryotic expression
polyclonal antibody
