Study On Fusion Expression Of Porcine Circovirus PCV3 Cap Protein And EGFP

Porcine circovirus type 3(PCV3)is a new type of porcine circovirus that was first discovered in the United States in 2016.It is highly invasive to pigs of all ages,especially piglets and sows during pregnancy are more likely to be infected.It is associated with diseases such as porcine dermatitis and nephropathy syndrome(PDNS),piglet congenital tremors(CT),sow reproductive disorders,and multisystem inflammatory response.Current research shows that PCV3 exists and is popular in many countries and regions in the world,so it is very important to detect and control PCV3.Current research shows that PCV3 has already existed and spread in many countries and regions in the world.Therefore,detection and prevention of PCV3 is very important.Based on the PCV3 Cap protein sequence and the codon bias of the E.coli a Cap protein gene in PCV3 was optimized and sythisized was linked with the EGFP fluorescent protein gene and inserted as the target gene between the Ncol and Hind? restriction sites of the pET28a vector to construct a pET28a-PCV3-Cap-EGFP recombinant plasmid.After identification by sequencing and PCR amplification,it was transformed into BL21(DE3),and the induction conditions such as IPTG concentration,induction time and induction temperature were optimized.According to the results of SDS-PAGE gel electrophoresis and laser scanning confocal microscopy analysis of the bacterial solution,it can be seen that the target protein with a size of 53.66 kDa was successfully expressed;the target protein was distributed in the supernatant and the precipitate,mostly in the form of inclusion body precipitation Existence;when induced at 1 mmol/L IPTG and 37? for 4 h,the expression of target protein is higher than others.The inclusion body precipitates were washed with inclusion body washing solution,8M urea dissolved,renatured with different concentration gradients,refolded and concentrated by ultrafiltration,and then observed by SDS-PAGE gel electrophoresis and laser scanning confocal microscope.The results showed that the target protein was successfully renatured.This experiment uses E.coli prokaryotic expression system to explore the fusion expression of PCV3 Cap protein and EGFP fluorescent protein,and lays a prerequisite for the development of PCV3-related diagnostic reagents and vaccines.