Porcine Circovirus Virus Type ? Prokaryotic Expression Of Cap Protein Purification And Enzyme Activity

Porcine circovirus virus(PCV)can be divided into ? porcine circovirus virus type 1(PCV)and porcine circovirus virus type ?(PCV 2).PCV 1 are sackless,PCV 2 is a major pathogen of porcine circovirus virus disease.PCV 2 gene has 11 major reading frame,including ORF2 reading frame coding of the surface of the Cap protein has a number of PCV 2 antigen epitope and the recognition sites,so Cap protein can be used as ideal porcine circovirus(PCV 2)virus antigen.Cap protein can serve as the advantages of porcine circovirus(PCV 2)virus antigen basically has the following points: 1.The composition is known,safe and reliable,non-toxic,no side effects;2.The protein content is controllable,the quality of the product unification between each batch.But in recent years,studies have shown that Cap protein expression mainly in the form of inclusion body,it's not easy to want to get Cap of soluble protein.Although there are research Cap can be made in the form of inclusion body protein after denaturation and renaturation of soluble protein can be obtained after processing.But the process is too complicated,and the process of denaturation and renaturation may affect the protein itself.So get soluble Cap protein both in for vaccine development and scientific research is extremely important.This experiment by building a recombinant prokaryotic expression vector p ET21b-MBP/EDA/GST/gamma crystallin-PCV2,we got soluble recombinant protein Cap.Cap protein found in the study of the protein is to identify and cut aspartic acid,histidine,a set of amino acids valine enzyme activity.1: build a recombinant vector p ET21b-MBP/EDA/GST/gamma-crystallin-PCV2.fter obtaining the use right of the PCV 2 gene by PCR and have the same enzyme loci four carrier p ET28-EDA/gamma-crystallin/GST/MBP construct recombinant protein EDA/gamma crystallin/GST(Glutathione S transferase)that Glutathione transferase/MBP(Maltose binding protein)-PCV 2 expression vector.We gained good soluble restructuring EDA/GST/MBP-Cap protein,EDA/GST-Cap protein soluble protein respectively accounted for 76.9% and 30.7% of the target protein.By sds-page analysis and Western blot,prove the recombinant plasmids in e.coli can be expressed protein and soluble farmers shell has good antigenicity.2: Cap protease activity.Experiment we found that cut Cap protein identification and aspartic acid,histidine,valine,a set of amino acids the enzyme activity.In order to further verify the Cap protease function,We put the carrier p ET21 b Spy Tagsf-GFP-Spy Catcher sf-GFP gene and Spy Catcher join point mutations can make translation egg Bai Tiandong amino acid,histidine,valine,a group of small peptides.Soluble Cap protein and mutation of Spy Tag-sf-GFP-Spy Catcher protein response.Sds-page gel again cut Cap protease can identify and aspartic acid,histidine,valine,a group of small peptides.Study on soluble Cap protein and mutation Spy Tag – sf-GFP-Spy Catcher protein reaction with belong to which kinds of enzyme inhibitors preliminary estimates Cap protein.Use software to analyze Cap protein secondary structure mutation PCV2 find Cap protein gene activity center.This research for the PCV 2 Cap protein function research,the mechanism of porcine circovirus virus infection and the development of subunit vaccine provides the basis.