Construction Of F-Cap Gene Expression Vector And Its Prokaryotic Expression

Porcine circovirus type 2(PCV2)is the primary causative agent of postweaning multisystemic wasting syndrome(PMWS)and other related diseases in pigs.PMWS has very high incidence,which has resulted in the huge economic loss to the pig industry.It was noted that vaccination is the most economical and most effective measure to prevent and control infectious disease.The available vaccines,can not prevent the spread of pathogens from the root.Ferritin widely exist in a variety of organisms,which function in regulating iron ion content in the body,to create a balance internal environment of iron ions for organisms.Since it can assemble into a shell nanostructures spontaneously and can be absorbed and degraded by the body,it is often used as drug delivery carrier,medical imaging diagnosis,nano biosensors,etc.In addition,the 24 subunit structure of ferritin can be self-assembly into nanoparticles through genetic modification and biochemical methods,a unified size and highly symmetrical features.Studies have shown that ferritin have a potential site for a variety of peptides and small insert protein antigens because of its internal carboxyl and external and ring structure,which can be used as a genetically introduce polypeptide antigen to its internal or external to the nanoparticles presented system.Given that so many characteristics of ferritin,we aim to verify the PCV and ferritin fusion expression,and explore the physical and chemical properties and biological activity of recombinant proteins.Firstly,the six victors with different fusion tags were chosen for the new recombinant plasmids by the comparison of nucleotide sequences between the multiple cloning site of the six victors and target gene.PCR and enzymatic digestion were used to get linear fragments of the target gene F-Cap and the vectors with different fusion tags,and then insert target gene into the vectors followed by transformed into the DH5?.Extracted plasmid from expanding culture of e.coli.The recombinant plasmids were determined by restriction enzyme digestion and DNA sequencing.After that transform the recombinant plasmid which have a right gene sequencing into BL21 and induced by IPTG.The expression of recombinant protein were identified by SDS-PAGE analysis.The bacteria which can express lots of recombinant protein were crushed by ultrasonic,and the supernatant was taken to analysis with SDS-PAGE electrophoresis.Finally,the Ni-NTA affinity Agarose was applied to purify the target protein.Protein affinity tags were removed by TEV enzymes for purpose.Purified proteins which were subjected to heat treat was observed with transmission electron microscope.The results altogether have shown that,relatively pure protein,was successfully obtained,which will lay a theoretical basis for the nanometer vaccine development.