Prokaryotic Expression, Antibody Preparation And Epitope Analysis Of Lipoprotein-Associated Phospholipase A2

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a key enzyme as a risk predictor involved in atherosclerosis and ischemic stroke, and belongs to group VIIA of the phospholipase. At present, the clinical diagnostic reagents of LP-PLA2 mainly are dependent upon importation from foreign countries. Then the cost required is expensive, which is limited the application of LP-PLA2 in china. Therefore, it is important for us to development the immunoassays diagnostic reagents of LP-PLA2.Objective The purpose of this study was to obtain human lipoprotein-associated phospholipase A2 protein and its antibody which was used to verify antigenic epitopes of LP-PLA2 predicted by bioinformatics. This study will afford a foundation for further establishing the immunoassays of LP-PLA2.Methods Total RNA was isolated from differentiated THP-1 cells, and LP-PLA2 gene was amplified by RT-PCR. The PCR fragments were then cloned into expression vector, following by sequencing analysis. The positive recombinant plasmid was transformed into E.coli BL21(DE3) and the recombinant protein LP-PLA2 was expressed, Then the purified protein was identified by SDS-PAGE and Western blot. Rabbits were immunized with the LP-PLA2 protein and the polyclonal antibody was obtained. At the same time, predicted epitope of LP-PLA2 using variety of bioinformatics methods, and the antigenicity was identified with the LP-PLA polyclonal antibodies.Results 1. LP-PLA2 gene was successfully cloned into Prokaryotic expression vector PET-28a(+)-LP-PLA2,PGEX-4T-2-LP-PLA2,and pCold TF-LP-PLA2.2. After expression induced by IPTG, SDS-PAGE results showed that there was no LP-PLA2 protein expressed in the PET-28a(+)-LP-PLA and a small quantities of LP-PLA2 recombination protein were found expressed in the PGEX-4T-2-LP-PLA2 existing in the form of inclusion bodies. Besides there was LP-PLA2 recombination protein expressed in the pCold TF-LP-PLA2,existing as soluble protein of E. coli.3. After LP-PLA2 expression supernatants were purified by Ni affinity chromatography and blue gel affinity chromatography respectively, the obtained LP-PLA2 protein which could reach a purity of about 90% was found to exist as a protein of molecular weight of about 45kD, and was identified by commercial polyclonal antibodies. reach 1:5.12×106 by ELISA. Then the specificity was confirmed by Western blot.5. Three epitope peptides of LP-PLA2 were predicted by bioinformatics software and were confirmed having good antigenicity by antigen-antibody immune response in vitro experiments. Conclusion The LP-PLA2 proteins and the specific polyclonal antibodies have been obtained. Three sections epitope peptides of LP-PLA2 were predicted by bioinformatics software and then confirmed of good antigenicity by antigen-antibody immune response in vitro experiments. What's more these peptides could be used for further preparing polyclonal antibodies and monoclonal antibodies. Thus this study could afford a foundation for further establishing the immunoassays of LP-PLA2.4. The polyclonal antibodies were obtained and titers examined could...