1种高效纳米抗体生产方法的建立  

Establishing a Method for Highly Efficient Producing Nanometer Antibodies

作　　者：贺生芳[1] 付向晶[1] 刘阳坤[1] 慕国辉[1] 刘海金[1] 王兴龙[1] 杜恩岐[1] 杨增岐[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2013年第9期1487-1493,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：西北农林科技大学引进人才科研启动基金(Z111021006)

摘　　要：为实现纳米抗体的简单、高效、低成本表达,在已有的商品化原核表达载体的基础上,对其进行改造,在靶蛋白N端分别融合3种不同串联融合标签His-Intein、His-Sumo-Intein和His-Trx-Intein,以本实验室筛选的抗猪圆环病毒2(PCV2)Cap蛋白的纳米抗体基因为靶蛋白,构建了pHSIE-Nb(His-Sumo-Intein)、pHSIE-intein-Nb(His-Intein)和pET32a-intein-Nb(His-Trx-Intein)3种表达载体。分别转化大肠杆菌BL21(DE3),用IPTG诱导融合蛋白表达,在不同表达条件下比较,选取可溶性表达最好的载体大量表达,用Ni-NTA金属螯合层析柱进行纯化,并利用Intein自剪切去除标签,纯化纳米抗体。且用制备的纳米抗体作为包被抗体,做双抗体夹心ELISA检测其反应原性。结果显示3种不同融合标签的纳米抗体都可成功表达,而融合His-Trx-Intein标签的载体可溶性表达量最高。融合蛋白经pH7、25℃剪切24h后可以得到不含融合标签的纳米抗体。双抗体夹心ELISA检测,统计学分析,制备的抗PCV2Cap纳米抗体蛋白包被的样品组与阳性对照组差异不显著(P>0.05),因此制备的纳米抗体是有活性的。本研究成功建立一种高效表达、纯化纳米抗体的方法,可以增加蛋白表达量、可溶性和简化蛋白纯化过程,获得具有活性的无标签的纳米抗体。为其进一步的纳米抗体理论研究和生产应用提供技术支持。The aim of this study was to establish a simple, efficient and inexpensive strategy for expression and purification of Non-taged nanobodies. Chose nanobodies of porcine circovirus type 2 （PCV2） Cap protein as the target protein, three plasmids with N-terminus fusion tags pHSIE- Nb（ His-Sumo-Intein）, pHSIE-intein-Nb（His-Intein） and pET32a-intein-Nb（His-Trx-Intein） were constructed and their effects were compared. The recombinant three plasmids were transformed into E. coil BL21（＋） and induced by IPTG respectively. The expression and solubility of target proteins were tested under different conditions, and the favorable tags were selected. Recombi- nant proteins were purified by Ni-NTA column chromatography, and tags were removed by Intein as self-cleavaged, then target protein were prepared, based on which a double antibody sandwich ELISA method was developed. All these three different tagged nanobodies could be successfully expressed. A series of experiments lead to the finding that the placement of His-Trx-Intein before the Nb is the best strategy in availing the soluble expression of the tagged protein. After on-col- umn cleavage was preceded at pHT. 0,being incubated at 25 ℃ for 24 h, the recombinant un- tagged Nb were released. The results of double antibody sandwich ELISA showed that the Nbprotein could react specifically with cap protein, and there was no significant difference （P〈0.05） between positive control group and nanobody group, which suggested that this method of preparation of nanobodies was successful. An efficient expression and simple purification method of preparation of nanobodies was successfully established, which was efficient soluble expression, and simple purification, and no tagged active nanobodies could be prepared. The study provides a technical support for the further theoretical research and production in nanobodies.

关 键 词：纳米抗体 融合标签 自剪切Intein 表达纯化 双抗夹心ELISA 

分 类 号：Q789[生物学—分子生物学]