摘要
目的制备a-N-乙酰半乳糖胺酶(NAGA)的高效价高特异性的兔多克隆抗体。方法利用pET22b-NA-GA/BL21(DE3)工程菌株表达NAGA,经Ni2+Sepharose 6 FF亲合层析,Phenyl Sepharose 6 FF疏水层析两步纯化,得到纯度>95%的NAGA作为抗原免疫新西兰白兔,获得NAGA的兔抗血清,并经HiTrap rProtein A柱纯化获得高效价高特异性的抗体;用间接ELISA法检测抗体效价,用Western blotting评价抗体特异性。结果制备得到的NAGA兔多克隆抗体血清效价为1×106,经过rProtein A柱纯化后抗体蛋白纯度>95%,效价为1×105;Western blotting显示:该NAGA兔多克隆抗体只与NAGA发生特异性反应。结论获得了NAGA的高效价高特异性的兔多克隆抗体。
Objective To prepare the high titer and high specificity rabbit polyclonal antibody of a-N-acetylgalac- tosaminidase（NAGA）. Methods The recombinant NAGA from Elizabethkingia meningosepticum was expressed in Ecoli. BI21 （DF-3）. Then, the expression products in supernatant were purified by Ni2＋ Sepharose 6 FF affinity chromatography and Phenyl Sepharose 6 FF hydrophobic interaction chromatography. The purification process resulted in greater than 95% pure product. The New Zealand rabbits were immunized with purified recombinant NAGA. Rabbit antiserum was purified by Hi- Trap rProtein A column. Its titer and specificity was detected by indirect ELISA and western blotting respectively. Results The titer of rabbit sera was 1×10^6. And the titer of antibody protein was 1×10^5 ,the purity was about 95% after the rProtem A purifying. Western blotting shows that the antibody protein reacted with NAGA only. Conclusion This kind of methods can obtain the high titer and high specificity rabbit polyclonal antibody of NAGA.
出处
《中国输血杂志》
CAS
CSCD
北大核心
2012年第7期652-654,共3页
Chinese Journal of Blood Transfusion
基金
国家自然科学基金(30801063)
