质粒pRSET-A前导肽串联多聚体的构建及其多克隆抗体制备*

质粒pRSET-A是一个常用的高效原核表达载体，编码一N端含组氨酸标签（6×His）的34aa前导肽序列，以方便利用抗组氨酸标签抗体鉴定或纯化所表达的重组蛋白。本实验设计一对两侧含编码疏水性氨基酸密码子的引物，经过扩增前导序列10~34aa基因序列，并重新克隆入质粒pRSET-A构建串联二聚体后，再利用质粒pRSET-A的BamH I / Bgl II同尾酶克隆位点，经一系列简单的酶切和连接，快速构建这一前导肽中不含组氨酸标签序列的串联多聚体基因，并成功表达其六聚体重组蛋白。将此重组蛋白主动免疫山羊，获得了能够特异地识别pRSET-A编码的N端前导肽序列的抗体。结果显示，所制备的羊抗10~34aa前导肽抗体能够识别pRSET-A指导表达的含有完整前导肽的重组蛋白，但不能识别不含10~34aa序列的重组蛋白；同时，利用同位酶技术可以快速高效构建短肽的串联多聚体以制备具有高免疫原性的亚单位疫苗或免疫调控物质。

Construction of Tandem Fragment Polymer to Generate Polyclonal Antibody for Recognizing Plasmid PRSET-A expressed Peptides

The plasmid pRSET-A, a common prokaryotic expression vector, encodes a N-terminal 34aa precursor peptide with a histidine tag sequence. Anti-histidine-tag antibody is usually used for identification of expression of the recombinant protein expressed using pRSET-A. A pair of primers that contained flanking hydropathic amino acid codons were synthesized, to amply the sequence coding for 10~34 aa in the precursor sequence. The amplified fragment was inserted into pRSET-A to generate the first double repeat of the precursor gene. By utilizing a pair of isocaudamer BamH I and Bgl II sites, and another downstream Hind III site of plasmid pRSET-A, following a series of simple double digestions and ligation of the resulted products, a series of repeat (3, 4 and 6) precursor peptide fragment genes were derived. The 6 repeat polymer of the precursor peptide was successfully expressed, and used to immunize goats for preparation of anti-precursor peptide antibodies. The antibody produced effectively identified the recombinant protein with the completed precursor peptide, but not the recombinant protein only had the His-tag sequence and lack the 10~34 aa sequence. These results demonstrated the repeated short-peptides polymer can be quickly and efficiently constructed by utilizing a pair isocaudamer sites in production of subunit-vaccine or immune-regulating antigens.