Construction of Tandem Fragment Polymer to Generate Polyclonal Antibody for Recognizing Plasmid PRSET-A expressed Peptides
质粒pRSET-A前导肽串联多聚体的构建及其多克隆抗体制备*

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.