| Swinepox(SWP)is a swine infectious disease characterized by swinepox virus(SWP V)causing skin damage,formation of pox and scarring,and most of the swine can recover spontaneously after infection.The damage to the pig industry is minimal.Because of its ch aracteristics of strict host specificity,large genome size(146Kb),multiple non-essential re gions(NER),and large insertable exogenous genes,swinepox virus is especially suitable for the genetic engineering vaccine carrier.Although SWPV has an attractive potential application as a vaccine vector,it remains to be affirmed whetherSWPV can be used as a vaccine vector for clinical application: screening of non essential regions(as exogenous gene insertion sites),screening of strong promoters,identification of virulence related genes and so on.This study with enhanced green fluorescent protein(EGFP)expression quantity as SWPV promoter strength the judgement index,all possible promoter in SWPV genome filtered(146)one by one,in the hope to find SWPV strong promoter,and to build for the future efficiently expressing of foreign protein SWPV vector system research providing strong promoter.1.Expression of enhanced green fluorescent protein(EGFP)Construct the recombinant expression vector pET-32a-EGFP-His by the prokaryotic e xpression vector pET-32 a.The recombinant protein EGFP-His was successfully expressed,and the recombinant protein was present in the form of inclusion body.The purified recombinant protein is colorless under visible light.SDS-PAGE results showed that the recombinant protein EGFP-His was about 48 k Da.The recombinant swinepox virus rSWPV-P28-EGFP-His was obtained by taking the SWPV-JX20 G isolate of Jiangxi Province as the parent virus,and using the TK gene as the insertion site of exogenous gene,and treating the promoter of vaccinia virus(VV)P28 as t he promoter of the foreign gene to construct the recombinant transfer vector pSW-P28-EG FP-His,and the recombinant swine pox virus rSWPV-P28-EGFP-His expressing EGFP was obtained by homologous recombination technology.The PK15 cells infected with recombinant swinepox virus rSWPV-P28-EGFP-His showed green fluorescence under fluorescent microscope,and the purified recombinant protein appeared green under visible light.The results of SDS-PAGE showed that the molecular weight of EGFP-His protein was about 27 KDa.2.Preparation of anti-EGFP monoclonal antibodiesUsing EGFP-His as immune antigen expressed by eukaryotic expression vector(SWPV vector)to immunize BALB/c mice.Using EGFP-His expressed in eukaryotic cells and EGFP-His expressed in prokaryotic cells respectively as detectable antigens.Using hybridoma cells technique and screened 9 strains of hybridoma cells,which could secrete anti EGFP monoclonal antibodies,were named 4B3,11H6,7E12,9H8,17C8,2F2,9A10,18A6 and 17H2,respectively.The experimental results of ELISA and Western blotting showed that 7 strains(2F2,4B3,11H6,9H8,17C8,18A6,17H2)could specifically react with the EGFP-His which the eukaryotic or prokaryotic expressed,and the other two strains(7E12 and 9A10)only reacted with eukaryotic expressed EGFP-His,and did not react with prokaryotic expressed EGFP-His.It can be speculated that the epitope of 7E12 and 9A10 monoclonal antibody are the EGFP conformation epitope,and the other 7Mc Abs are EGFP linear epitope.3.Screening of strong promoter of pig pox virusThe swinepox virus genome contains 150 open reading frames(ORFs,SWPV001～SWPV150).Since SWPV001～SWPV004 and SWPV147～SWPV150 are complementary to each other,there are 146 promoters of SWPV.Based on the whole genome sequence of swinepox virus Jiangxi isolate(SWPV-JX20G),146 primers for PCR amplification of promoter(Px)were designed.Taking SWPV-JX20 G strain was used as the parent virus,and TK gene as the as the insertion site,EGFP as the reporter gene and the promoter Px of swinepox virus as the promoter of the reporter gene,146 Px-EGFP containing transfer vectors were constructed.146 recombinant viruses rSWPV-Px-EGFP were obtained by homologous recombination technology.According to the fluorescence brightness and Western-bloting results of recombinant swinepox virus rSWPV-Px-EGFP infected PK15 cells,the strength of the selected swinepox virus promoter was determined,and the results of this experiment showed that the 146 swinepox virus promoters has great different effects,and they could be divided into three categories:(1)the 6 promoters that are more effective than P11 are: P026、P033、P067、P069、P109、P150;(2)8 promoters which have few differences between the starting effects of P11: P006、P025、P027、P055、P060、P120、P145、P147;(3)the other promoters that are extremely less effective: P010、P040、P112、P117、P119、P135、P139、P148.Up to now,there has no identification of the promoter of swinepox virus at domestic or foreign literature reports.This study screened all the promoters of swinepox virus genome(146),and had screened 6 strong promoters(P026、P033、P067、P069、P109、P150),and built the foundation for the future research on the construction of a high-level expression vector for swinepox virus. |
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