高免疫原性骨桥蛋白的构建、表达及鉴定

Construction and expression of high immunogenic osteopontin fusion proteins

目的重组及表达高免疫原性骨桥蛋白（OPN）的融合蛋白。方法使用Lasergene软件分析OPN基因，选取出免疫原性高的区域作为目的片段。提取总RNA，应用逆转录、PCR等技术扩增OPN基因目的片段，将其分别与带有His标签的pET-32a及GST标签的pGEX-4T-1载体连接。构建表达载体，诱导表达OPN融合蛋白（分别含有His，GST—Tag），并进行SDS—PAGE及Western blotting鉴定。结果扩增后获得一条351bp的DNA片段，经测序鉴定为目的片段序列，并实现了可溶性高表达，经Western blotting鉴定为高免疫原性OPN融合蛋白。结论采用原核表达经纯化后可获得高纯度高免疫原性的OPN融合蛋白，为进一步开发OPN诊断试剂打下基础。

Objective To clone and express osteopontin（OPN）fusion proteins with high immunogenic-ity. Methods OPN gene was analyzed using Lasergene software, and a high immunogenic region was selected as target fragment. The fragment of the target gene was cloned by RT-PCR from total RNA, The amplified gene was inserted into vectors pET-32a and pGEX-4T-1. The recombinant plasmids were constructed and expressed proteins （including His-Tag and GST-Tag, respectively） were identified by SDS-PAGE and Western blotting. Results A DNA band about 351 bp was confirmed as target OPN fragment by DNA sequencing. Sohlble ex-pression of high immunogenic OPN fusion proteins was achieved, which was identified by Western blotting. Con-clusion High immunogenic OPN fusion proteins with high purity have been expressed successfully in prokary-otic system. The study lays a foundation for further development of OPN diagnostic reagent.