Construction And Identification Of Recombinant Encoding PrME Protein Derived From Japanese Encephalitis Virus And ImmunoglobulinG Fc Of BALB/c Murine

ObjectiveIn the present study,we got gene encoding immunoglobulin G Fc from BALB/c murine spleen cells by nested-RT-PCR technique,gene encoding JEV prME protein was obtained with restriction endonuclease BamHI/EcoRI from the eukaryotic recombinant named after pJME,which was constructed by us before.We constructed the recombinant with fusion gene encoding prME protein and immunoglobulin G Fc by putting them into a eukaryotic expression vector pcDNA3.1(+).The recombinant was named pJME/IgG Fc and CHO celluar lines expressed fusion protein encoded by fusion gene stably were obtained.It would establish ground for reinforcement effect study of Japanese encephalitis DNA vaccine.Materials and Methods一.Materials1.Cells and animalsChinese hamster ovary cells were purchased from shanghai cell bank of chinese academy of science and grown at 37℃in Dulbecco' modified eagle medium(D-MEM) supplemented with 10%fatal calf serum(FCS)and 5%CO₂.BALB/c murine were purchased from shanghai animal center of chinese academy of science.2.plasmids and strainsThe recombinant with gene encoding prME was constructed by us before and named pJME,eukaryotic expression vector pcDNA3.1(+)was purchased from Invitrogen company,which was used for construction of fusion gene encoding JEV prME protein and IgG Fc.Escherichia coli JM109 and DH5αwere purchased from Takara company and were used for construction and transformation of recombinant plasmid.3.main reagentsRNAiso Reagent(Code No.D312),High Fidelity PrimeScript^TMRT-PCR Kit(Code No.DR027A),DNA Fragment Purification Kit Ver.2.0(Code No.DVS07),DNA ATailing Kit(Code No.D404),Agarose Gel DNA Purification Kit Ver.2.0(Code No.DV 805),DNA Ligation Kit＜Mighty Mix＞(Code No.D6023),Plasmid Minidose Abstraction Kit(Takara,product No.DV801A),restriction endonuclease EcoRI,BamHI and NotI were all purchased from Takara company,which were used construction and identification of recombinant plasmid.Lipofectamine2000 was purchased from Invitrogen company and was used for transfection into CHO cells.G418 was purchased from GiBCO company and used for screening of resistant cloned strain.Ampicillin was puchased from Sigma company,Agarose was purchased from Takara company,they were all used for transformation and electrophoretic analysis of plasmid.Dulbecco' modified eagle medium,10%fatal calf serum,penicillin and streptomycin were all purchased from Hyclone company and used for cultivation of CHO cells. Goat-Anti-Mouse IgG Fc was purchased from Santa company,FITC-Rat-Anti-Goat CD32 was purchased from beijing Zhongshan company,they were all used for immunofluorescence.Protein lysate,loading buffer,PVDF membrane,prestained protein Marker were all purchased from biyuntian company,HRP-Rat-Anti-Goat IgG were all purchased from beijing Zhongshan company and used for Western-blot analysis.二.Methods1.The construction of recombinant to express Japanese encephalitis virus prME protein and IgG FcTotal RNA was isolated from BALB/c murine spleen cells.The cDNA fragment of IgG Fc was generated by nested-RT-PCR amplification of genomic RNA and was modified by plusing a "A" tail and Gly-linker.It was constructed into EcoRI/NotI site of the vector for sequencing named pMD19-T simple and was sequenced by an ABIPRSM^TM310Genetic Analyzer(Pekin-Elmer/Applied)and Bca BEST Primer RV-M, and compared to the published IgG Fc CDS region sequence by Genebank.Reverse transcription primer and 3'-outer primer:5'GGT GGA GGT AGG TGT CAG AG 3', complementing to nucleotides(nt)1491 to 1510 in the IgG Fc CDS region.3'-intra primer:5' GCG GCC GCT TTA CCA GGA GAG TGG GAG AG 3',complementing to nt1432～1460 in the IgG Fc CDS region.5'-outer primer:5'AGC GAG ACC GTC ACC TGC AA 3',hybridizing to nt709 to 728 in the IgG Fc CDS region,5'-intra primer:5'GAA TTC GGT GGA GGC GGT TCA GGT GGA GGT GGT TCA GGA GGA GGT GGA TCG ATG GTG CCC AGG GAT TGT GGT TGT AAG 3', hybridizing to nt718 to 795 in the IgG Fc CDS region.For cloning of IgG Fc genes, restriction enzyme site for EcoRI(-GAA TTC-)was engineered at the 5' terminus and NotI(-GCG GCC GC-)was engineered at the 3' terminus.RT-PCR-amplified product of IgG Fc was digested with restriction enzymes EcoRI and NotI,and was inserted into the pcDNA3.1(+)vector at EcoRI/NotI site.The construct containing IgG Fc region was designated pcDNA3.1(+)/IgG Fc.Competent Escherichia coli DH5αcells was transformed with pcDNA3.1(+)/IgG Fc and plated on Luria Broth(LB)agar plates that contained ampicillin(100mg/L,sigma).Clonies was picked up and inoculated into 3mL LB medium containing ampicillin(100mg/L).PcDNA3.1(+)/IgG Fc was extracted from large scale culture by minidose abstraction kit.It was identificated with restriction enzyme EcoRI/NotI and sequenced similarly.Competent Escherichia coli DH5αcells was transformed with recombinant pJME and it was minidose abstracted similarly.It was identificated with restriction enzyme BamHI/EcoRI and was sequenced similarly. Plasmid pcDNA3.1(+)/IgG Fc was digested with restriction enzymes BamHI/EcoRI, and gene encoding prME was inserted into its BamHI/EcoRI site,so the recombinant plasmid to express JEV prME protein and IgG Fc was constructed and named pJME /IgG Fc.Competent Escherichia coli DH5αcells were transformed with pJME/IgG Fc, then pJME/IgG Fc were minidose abstracted and idenificated with restriction enzyme BamHI/EcoRI,BamHI/NotI.2.pJME/IgG Fc was transfected into CHO cells to get CHO cell lines which express fusion proteins stablyDetermined optimal screening concentration for our experiment:CHO cells(1000 cells/mL)had been incubated in the 24-well format(0.7mL/wel1)for 6 hours,then G418 with different concentration were added into every well,such as 600mg/L, 650mg/L,700mg/L,750mg/L,800mg/L,850mg/L,900mg/L,each concentration had three wells.The last three wells were CHO cells untansfected.Following incubated for 11 days,the lowest concentration which killed all CHO cells was the optimal screening concentration,It was 800mg/L.PJME/IgG Fc was transfected into CHO cells by the liposome method,at the same time,CHO cells transfected with pcDNA3.1(+) and CHO cells untransfected were used as negative controls.One day before transfection, 4×10⁵ CHO cells were plated in 500μl of D-MEM without antibiotics per well so that they would be 90-95%confluent at the time of transfection.For each transfection sample,DNA-Lipofectamine 2000 complexes were prepared as follows:a.DNA(0.8μg) were diluted in 50μl of D-MEM without serum then mixed gently,b.2μl Lipofectamine 2000 were diluted in 50μl of D-MEM without serum.Mixed gently and incubated for 5 minutes at room temperature,c.After the 5 minute incubation,the diluted DNA with the diluted Lipofectamine 2000(total volume was 100μl)were combined and had been incubated for 20 minutes at room temperature to allow the DNA-Lipofectamine 2000 complexes to form.100μl of DNA-Lipofectamine 2000 complexes were added to each well containing cells which had been prewashed twice with 1mL of D-MEM without FCS.The cells had been incubated at 37℃in a CO₂ incubator for 6 hours,then medium was replaced with D-MEM with 10%FCS and incubated for another 18 hours. The cells were passaged at a 1:10 into fresh growth medium 24 hours after transfection. Selective medium was added with 800mg/L G418 the following day to screening.The medium was replaced every three days according to the colour of medium,then medium with 800mg/L G418 was replaced with 400mg/L G418 after seven days to magnify the masculine clone.3.The fusion proteins encoded by pJME/IgG Fc gene in transfecd CHO cells were detected by immunofluorescenceCHO cells were plated in a 6-well format,when they were 60-70%confluent,4% paraformaldehyde had been added into each well containing CHO cells for 1hour, 0.1%Triton-100 for 10min,10%BSA for 1 hour,then they had been incubated with Goat-Anti-Mouse IgG Fc for 18 hours and with FITC - Rat - Anti - Goat CD32 for 1 hour at 37℃without light.4.Western blot analysis Cellular lysates were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis[SDS-PAGE(8%polyacrylamide)],and transferred onto a PVDF membrane in transfer buffer for overnight at 60V.Then the membrane had been incubated in 5%defatted milk powder for 1 hour to blocking and with Goat-Anti-Mouse IgG Fc for 2 hours at room temprature.Following thrice of 10min washing with TBST buffer,the membrane had been incubated with HRP-Rat-Anti-Goat IgG for 1 hour at room temprature. After washing with TBST,the fusion protein encoded by pJME/IgG Fc was detected by Diamino benzidine(DAB)staining method.Results1.Construction and identification of the recombinant pJME/IgG FcMolecular weights(2001bps,2730bps)of the two inserts released from pJME/IgG Fc with two groups of restriction analysis associated with BamHI/EcoRI and BamHI/NotI were correlated to the expected theoretic results respectively.The sequence analysis showed that two genes were in their corret sites.2.Transfection of pJME/IgG Fc in CHO cellsOptimal screening concentration of 6418 was 800mg/L.CHO cells transfected with pJME/IgG Fc formed several celluler thyses after incubated with 800mg/L 6418 for 7 days,and they growed up and fused gradually during incubated with 400mg/L 6418 for 26 days.3.Fusion protein was detected by immunofluorescenceThe green fluorescence mark was mainly distributed obviously in endochylema of transfected CHO cells with pJME/IgG Fc,and not much in membrane.It didn't apperance in untransfected CHO cells and transfected CHO cells with pcDNA3.1(+). Sometimes,some unspecificial fluorescence substance apperanced in them.4.Fusion protein was detected by western-blotA single protein band was detected at Mr＞95kD region in CHO cells transfected with pJME/IgG Fc,It didn't apperance in untransfected CHO cells and transfected CHO cells with pcDNA3.1(+).Conclusions1.The recombinant named pJME/IgG Fc contained genes of prME and IgG Fc. 2.CHO cells transfected with pJME/IgG Fc could express prME and IgG Fc fusion protein stably.3.The expression of above fusion protein was mainly distributed in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells.It was estimated that molecular weight of the fusion protein was101KD.