In-vitro Expression Of Proliferating Cell Nuclear Antigen And Production Of Polyclonal Antibody Against PCNA From Prorocentrum Donghaiense

In this study, Prorocentrum donghaiense and Skeletonema costatum, two domain species of red tide algae in East China Sea area, were taken as the objects. Proliferating cell nuclear antigen (PCNA) of two algaes are expressed in vitro and tested immunization effects. The aim is to lay good foundation in development of red tide algae.The gene coding for proliferating cell nuclear antigen (PCNA) in Prorocentrum donghaiense and Skeletonema costatum were sequenced and transcript to amino acid sequences using DNAStar searching Open Reading Frame. Compare the two species'PCNA amino acid sequences, select the high specificity region as the target area and then transcript these sequences into gene sequences according to optimal coding theory. The fragment of PCNA gene of Prorocentrum donghaiense and Skeletonema costatum were synthesized. The synthese gene was inserted into PUC57 plasmid and then transformed into bacteria host strain DH5α. After colony culturing and screening, a recombinant plasmid was obtained. Plasmids were double digested and gene fragments were inserted into pGEX-6P-1 vectors and transformed into BL21 bacteria strain. Screening the monoclonal strains which contain target gene to culture, add IPTG to induce gene expression. The recombinant subunit was purified by means of sonitation, GST affinity chromatography, and then the obtained protein was examined by SDS-PAGE. The result shows that PCNA of these two species was successfully expressed and the purity was more than 85%, so it was pure enough as antigen.Based on the above work, taking Prorocentrum donghaiense as the objects, we carried out further studies on the method of preparing polyclonal antibody against PCNA. The protein fragment of PCNA was then mixed with Freund's adjuvant to immune mice. After 4 times routine immunization and 4 times supplementary immunization, extract blood serum from immuned mice's tails to indirect ELISA detection which shows the polyclonal antibody against PCNA of Prorocentrum donghaiense was successfully obtained and its titer is higher than 80000, so it can be used in preparing the monoclonal antibody...