Preliminary Exploration On JSRV-Env Protein Interact With Sheep Hyaluronidase2

Background:Jaagsiekte sheep retrovirus (JSRV), a type of β retrovirus,causes ovine pulmonary adenomatosis (OPA).JSRV-env gene mainly codes envelope protein (Env) which including two subunits:surface protein (SU) and transmembrane protein (TM). Recent studies have shown that sheep hyaluronidase2(Hyal-2) plays a role as receptor of JSRV and endogenousJSRV in sheep. Up to now the research data of interaction between sheep Hyal-2and JSRV envelope protein (its subunits) is still rather scarce.Objective:To study the relationship of JSRV-Env(SU and TM) and sheep Hyal-2further, in order to deeply understand the moleculer pathogenesis of OPA.Methords:(1) To expand the sheep hyal-2gene by using overlapping PCR and to predict the structure of its coding protein Hyal-2by bioinformatics software.(2) To construct the eukaryotic expression vector of sheep hyal-2gene for establishing the stable cell line expressing sheep Hyal-2.(3) To construct the eukaryotic expression vector of JSRV-Env (SU and TM) and verify the effect by transient transfection and sequencing analysis. To determine the localization of JSRV-Env (SU and TM) within cells by laser confocal microscopy. To test the combine of Env with Hyal-2by using co-immunoprecipitation and bimolecular fluorescence complementation system.(4) To amplify JSRV-sw defective gene by overlapping PCR and construct a series of eukaryotic expression vector for preparing SU defective proteins. To verify the success by transient transfection andsequencing analysis. To determine the localization of JSRV-SU within cells by laser confocal microscopy. To predict the possible combining domains between JSRV-SU and Hyal-2by bimolecular fluorescence complementation system and co-immunoprecipitation method.(5) To detect the expression changes of pik3ca and ras genes in mouse NIH3T3cells which were transiently transfected alone into the eukaryotic vectors of pEGFP-C1-(env、su、tm). To determine the expression changes of pik3ca and ras genes in mouse N1H3T3cells which the eukaryotic expression vectors pEGFP-C1-(env、su、tm) and pDsRed-monomer-N1-hyal-2were co-transfected together.Results:(1)The hyal-2gene has been expanded by overlapping PCR and its structure has been predicted by bioinformatics software.(2)The eukaryotic expression vector of Sheep Hyal-2was successfully constructed carrying a reporter gene of red fluorescent protein.(3) It has been verified that sheep Hyal-2combined with Env and SU protein by using co-immunoprecipitation and bimolecular fluorescence complementation system. It has been confirmed co-localization of JSRV-Env, SU with Hyal-2, respectively; there is no co-localization between JSRV-TM and Hyal-2.(4) A series of eukaryotic expression vectors expressing defective SU proteins have been successfully constructed, and these vectors could transient expression in eukaryotic cells.It has been confirmed co-localization of defective SU proteins respectively with Hyal-2by using confocal laser scanning method; The possible binding domains of SU interact with Hyal-2have been confirmed by co-immunoprecipitation and bimolecular fluorescence complementation system. The results showed that defective SU1-SU5proteins are not binding with sheep Hyal-2and the missing areas of SU1-SU5were very important for interaction between Hyal-2and Env protein. This result will be helpful for further determination of binding sites of sheep Hyal-2with SU protein.(5) In mouse NIH3T3cells which were transfected by pEGFP C1-(env、su、tm) vetors alone, the expression quantity of pik3ca and ras genes increased significantly compared with control group.Whereas, NIH3T3cells were co-transfected pEGFP C1-(env、su、tm) and pDsRed-monomer-N1-hyal-2together, the expression quantity of pik3ca and ras genes decreased significantly.Conclusions:(1) The hyal-2gene has been expanded by overlapping PCR and Hyal-2structure has been predicted by bioinformatics software.Eukaryotic expression vector of hyal-2was successfully constructed and transfected into293T cells. Hyal-2is a kind of hydrophilic protein and54.190kD of molecular weight.(2) Eukaryotic expression vectors of JSRV-Env protein and its subunits have been constructed and expressed in293T cells.(3) pEGFP C1-(env、su、tm) vetors and pDsRed-monomer-N1-hyal-2have been co-transfected into293T cells and they were co-expressed in293T cells.Either JSRV-Env or SU interacted with Hyal2,but there is no interaction between JSRV-TM and Hyal-2.(4) The expression products of defective sul-su5genes affect combination with Hyal-2protein,and238bp-867bp domain of su gene is necessary for the combination.(5) The expression quantity of pik3ca and ras genes in mouse NIH3T3cells which were transfected by pEGFP C1-(env、su、tm) vetors alone increased significantly compared with control group.The expression quantity of pik3ca and ras genes in mouse NIH3T3cells which were co-transfected by pEGFP C1-(env、su、tm) and pDsRed-monomer-Nl-hyal-2decreased significantly compared with control group.