| Equine Viral Arteritis (EVA) can cause prominent economic losses for the equineindustry. The purpose of this study is to develop some diagnosis technique by molecularbiology, virology, immunology etc. Major envelope glycoprotein(GP5 or GL),membraneprotein (M) and nucleocapsid(N) are three of the most immunogenic viral structuralproteins of equine arteritis Virus. They are amplified by RT-PCR from virus genome RNA.The PCR products were directly cloned into the clone vector pUC18 or pMD18-T vector,pUC18-GP5, pUC18-M and pMD18-T-N were obtained. Their sequences are sequencedand analysis by DNAstar software. The prokaryotic vector pET32a and pGEX-6p-1 wereemployed to construction of GP5, M, N and truncated M genes (named Mt). Theserecombinant plasmids, pET32a-GP5, pET32a-M, pET32a-N, pGEX-6p-1-GP5,pGEX-6p-1-M, pGEX-6p-1-N and pGEX-6p-1-Mt were constructed and were induced byIPTG, Their expression products were identified by SDS-PAGE and Western Blot.Indirect ELISA method was developed on the basis of their expression products. TheHansenula polymorpha yeast expression vectors were constructed by pHFMDHZ-α-Ashuttle plasmid, They were named pHFMDHZ-α-GP5, pHFMDHZ-α-M, pHFMDHZ-α-N,respectively. The recombinant Hansenula polymorpha containing GP5, M, N gene ofEAV were obtained. According to sequence of GP5, M, N protein of EAV, multi-epitopegene were designed agree with optimized codons in Hansenula polymorphayeast.GP5-M-N modified gene was synthesized by overlap PCR. The recombinantHansenula polymorpha containing GP5-M-N gene were obtained and induced by 0.5%methanol. The eukaryotic plasmid(mammalian), pVAX1-GP5, pVAX1-M, pVAX1-N andpcDNA3-GP5, pcDNA3-M, pcDNA3-N were constructed, respectively. BHK-21 cellswere transfected by these plasmids via transfection reagent. Rats were inoculated withthese plasmids. via intramuscular injection. In addition, Taqman RT-PCR was establishedand compare with conventional RT-PCR. This paper content as follows:1 Three pairs of primers for RT-PCR were designed according to the sequencesfrom GenBank to amplify major envelope protein gene, membrane protein gene andNucleocapsid gene of equine arteritis virus. The amplicons were cloned subsequently intothe pUC18 or pMD18-T easy plasmid. The recombinant plasmids pUC18-GP5, pUC18-Mand pMD18-T-N were identified by restriction enzyme digestion and sequencing.Nucleotide sequences analysis showed that the major envelope protein gene contains anopen reading frame of 768 bp, encoding a 255 aa protein, the membrane protein containsan open reading frame of 489 bp, encoding a 162 aa protein and Nucleocapsid genecontains an open reading frame of 333 bp, encoding a 110 aa protein, respectively. Thecomparison of the three genes with that of EAV NC-002532 strain showed that homologyof nucleotide sequence were 99.1% ,99.4% and 98.8%,respectively;and homology ofdeduced amino acids were 96.9% , 98.2% and 99.9%,respectively. The results showedthat the virus is belong to American virus and phylogenesis is near with prevalence virusin epidemiology.The prokaryotic expression plasmids containing GP5, M , N and truncated M gene ofEAV were constructed, named, pET32a-GP5, pET32a-M, pET32a-N, pGEX-6p-1-GP5,pGEX-6p-1-M, pGEX-6p-1-N and pGEX-6p-1-Mt, them were sequencing and verifiedtheir ORF and sequence. Then them were transformed into the host cell, E. coli,BL21(DE3) and the expression procedure was optimized including cultivated temperature,optional induction concentration and time of IPTG etc. The result indicated that thenucleocapsid protein gene either in pET32a-N plasmid or pGEX-6p-1-N plasmid can beexpressed efficiently with 0.8～1.0 mmol/L IPTG and 4 hour induction. The truncatedmembrane protein also can be expressed efficiently in the same condition. The expressionyields was amount to 50% of the total mass of bacterial entirely protein. These expressionproteins were identified by SDS-PAGE and Western blotting. The resulting Trx-Nrecombinant fusion protein was identified to be consisted of 34 kDa protein bands bySDS-PAGE and Western blotting analysis. The resulting GST-N recombinant fusionprotein was identified to be consisted of 40 kDa protein bands by SDS-PAGE and westernblotting analysis. The resulting GST-Mt recombinant fusion protein was identified to beconsisted of 34 kDa protein bands by SDS-PAGE and western blotting analysis. But TheE. coli containing GP5 and M gene were not detected their expression protein bySDS-PAGE and Western blotting. These results indicated that the recombinant fusionprotein, Trx-N, GST-N and GST-Mt, could be used as antigen of diagnostic assay fordetecting antibodies. 2 The yeast expression vectors containing GP5,M,N structural protein gene ofequine arteritis virus were constructed by secretion expression vector, pHFMDHZ-α-A.The recombinant Hansenula polymorpha containing interested gene were obtained. After96-120h induced by 0.5% methanol, These concentrated cultural solution supernatantwere purified by Co2+ resin. The results of SDS-PAGE and Western immnoblottingrevealed that GP5,M,N protein were not expressed in Hansenula polymorpha. Codonbias was one of the important factors which influence heterogenous gene expression,optimizing codon sequence could improve expression level of heterogenous gene.Because of wild-type genes were not expressed in Hansenula polymorpha, in order toimprove the expression level of gene in Hansenula polymorpha , according to the usageof gene codons in Hansenula polymorpha. GP5 protein peptide fragment including 46～53aa, 77～84aa, 203～213aa, M protein peptide fragment including 106～120aa, 145～156aa and N protein peptide fragment including 2～10aa, 16～24aa, 35～44aa wereselected and translated into neucletide DNA chain. The DNA chain, named, GP5-M-Nwas synthesized by overlap PCR and cloned into pMD18-T simple vector. The secretionexpression vector, pHFMDHZ-α-GP5-M-N, were constructed by subclone method. Therecombinant plasmid pHFMDHZ-α-GP5-M-N was transformed into Hansenulapolymorpha competent cells by electroporation. Positive clones were selected on YPDplates with Zeocine antibiotic and confirmed by PCR. The recombinant Hansenulapolymorpha containing GP5-M-N gene was screened and obtained. The SDS-PAGE andWestern Blotting analysis showed that the expression of GP5-M-N was achieved byrecombinant Hansenula polymorpha. It suggested the strategy to improve the expressionof gene in Hansenula polymorpha by codon optimization was successful. But expressionyield was lower than expectation. 3 To construct the eukaryotic expression vector of equine arteritis virus majorenvelope protein gene, membrane protein gene and neucleocapsid protein, and observe itsexpression in vitro as well as immune response were injected into rats by muscle route,the recombinant expression vector pVAX1-GP5, pVAX1-M, pVAX1-N andpcDNA3-GP5, pcDNA3-M, pcDNA3-GP5 were constructed by inserting the GP5, M andN gene into the eukaryotic expression vector pVAX1 and pcDNA3, respectively.Restriction enzymes digestion analysis and sequencing results revealed that theserecombinant expression vectors have been constructed successfully. All of these plasmidswere transfected into BHK-21 cells by lipofectamine reagent and the expressed productwas detected by indirect immunofluorescence. The indirect immunofluorescence resultshowed green fluorescence on the membrane of transfected cells. The result indicated thatthe constructed eukaryotic expression vectors, pVAX1-GP5, pVAX1-M, pVAX1-N andpcDNA3-GP5, pcDNA3-M, pcDNA3-GP5 can be expressed in vitro. IncompleteFrusends was chosen as adjuvant and mixed with the plasmids to form DNA vaccine.Eleven groups of rat were immunized with a dosage of 100 μL (0.5 g/L ) of plasmidspcDNA3-GP5, pcDNA3-M, pcDNA3-N, pcDNA3-GP5+pcDNA3-M+pcDNA3-N,pVAX1-GP5, pVAX1-M, pVAX1-N, pVAX1-GP5+ pVAX1-M+pVAX1-N , pVAX1,pcDNA3, and inactived EAV virus vaccine, respectively, for 4 times at 14 days intervals.Indirect ELISA and neutralization were employed to determine the immunologicalresponse. Results showed that: After 3 times of immunization, seroconversion occurred inall groups excepting negative controls. Antibody level went up swiftly 14 days after thethird injection of DNA vaccine. The immunological effect of DNA vaccines based on thepcDNA3 was entirely better than those based on the pVAX1. The antibody level themstimulated were significantly higher than the others. Among these monogene groups,pcDNA3-GP5 groups antibody was the highest than others, D mean value was 0.886, Theresult was significantly higher than pcDNA3-M and negative control group, but was notsignificantly than pcDNA3-N. The mean D value was 0.912 inpcDNA3-GP5+pcDNA3-M+pcDNA3-N combination groups. The results of pVAX1 allgroups were similarity to pcDNA3 all groups. Among pVAX1 monogene groups,pVAX1-GP5 group antibody was the higher than others, its mean D value was 0.523.Thehighest group was three monogene combination group, its mean D value was 0.553. Thetotal ELISA titer of antibodies in pVAX1 group was lower than pcDNA3 group. The SNtiter index of antibodies of results from pVAX1 all groups were similarity to pcDNA3 allgroups. The pcDNA3-GP5 group mean SN index was highest in monogene groups. Itsvalue was 3.28. The recombination group was 3.68. The pVAX1-GP5 group mean SNindex was highest in monogene groups. Its value was 2.62. The recombination group was3.0. The study provided fundamental data and materials for the preparation anddevelopment of DNA vaccine against EAV in horse. 4 A quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR)is developed for EAV detection by appropriate primers and probes were selectedaccording to the sequence of nucleocapsid protein gene of EAV. The method wasemployed to detect a series of dilutions cells virus containing different TCID50 fromBHK-21 and PRRSV. The method was compare with conventional RT-PCR and virusisolation. The quantitative TaqMan RT-PCR was found to be equal or superior to thereference methods. Reproducibility and sensitivity were tested and proved that the assaywas very reliable. Standard dilutions included in each test allowed absolute quantitative ofthe amount of viral RNA and could detect at least the virus of 10-TCID50.The TaqMan 4assay described is time-saving, easy to handle, exhibits a decreased risk ofcross-contamination and is highly sensitive and specific. It is, therefore, considered to bea powerful tool for the rapid detection and differentiation of EAV. This assay is ideal forthe study of EAV pathogenesis and persistence. 5 The recombinant plasmid pET32-N was transformed into E.coli BL21(DE3) hostcell and the expression products-recombinant nucleocapsid protein of EAV was obtainedunder the optimized condition of host cell cultivation and IPTG induction. Subsequently,the expression product was purified by the means of Co2+ resin protein purificationprocedure. Following SDS-PAGE and Western-blot were employed to detect thepurification effect and the specificity of purified recombinant nucleocapsid protein ofEAV. Then, the purified N protein was employed to be coated on the well of 96-wellplates, each following step was optimized, such as coating concentration of recombinantnucleocapsid protein, scample diluent and blocking solution etc. The findings indicatedthat recombinant nucleocapsid protein the most appropriate concentration was36.5μg/mL ,HRP-IgG goat anti rat most appropriate concentration of goat anti ratHRP-IgG was 1:400.The resulting N recombinant protein could not react with PRRSV... |
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