Sequencing And Analysis Of Ep-1 Like Gene Family Of Cotesia Vestalis (Hymenoptera: Braconidae) Bracovirus (CvBV) And Expression Of Ep-1 Like 1 Gene

Polydnavirus(PDV)is a kind of special insect viruses which shares a symbiotic and mutualistic relationship with some hymenoperan endoparasitiods.The expression of the PDV genes in the host is very important for disrupting host immunity and protecting the developing of endoparasitiods' eggs.EP gene family,which has very important physiological functions,is one of the most important gene families of bracoviruses.In this thesis,we sequenced and analysed 7 genes of the EP-1 like gene family of Cotesia vestalis bracovirus(CvBV),one of which,EP-1 like 1 gene(EPL-1), was then cloned into pET-28a and pET-30a prokaryotic expressive vectors,and the constructed recombinant plasmids pET28-EPL-1 were transformed into the host bacteria E.coli BL21.The expressive product induced by IPTG was identified by SDS-PAGE and Western Blot analysis.The produced protein was purified and then injected into New Zealand rabbit to induce immune serum(polyclonal antibody), which were used to analyse the expression dynamics of the EP-1 like proteins in the host.Furthermore,the EPL-1 gene transcription dynamics in the host Plutella xylostella larvae was analysed by quantitative real-time PCR.Main results are summarized as followed:(1)The full cDNA sequences of 7 EP-1 like genes of CvBV were obtained using Rapid Amplification of cDNA Ends(RACE)and their signal peptides,modification and fuctional sites were predicted using EXPASY tool and SignalP software,The results showed that there is no conserved domain but one signal peptide in each EP-1 like gene.There are a lot of modification and fuctional sites in EP-1 like gene family. For phylogenetic analysis,a distance tree was generated using PAUP~*4.0 from the alignment of the homologous protein sequences retrieved by BLASTP analysis using CvBV EPL as query.The result showed that there is no evident close homological relationships among the EP-1 like genes,which can be attributed to 4 different clades: (1)EPL-1 and EPL-2,(2)EPL-3 and EPL-4,(3)EPL-5 and CcBV early-expressed protein,and(4)EPL-7 and some GiBV and CcBV sequences.(2)The ORF of the CvBV EPL-1 gene was amplified by PCR;its size is 921bp. The gene was cloned into pET-28a and pET-30a prokaryotic expressive vectors,and the constructed recombinant plasmid pET28-EPL-1 was selected and transformed into the host bacteria E.coli BL21 DE3.The expressive product induced by IPTG was identified by SDS-PAGE and Western Blot analysis.The results showed that the recombinant vector pET28-EPL-1 produced a 34.8kDa inclusion body protein,which was purified and then injected into New Zealand rabbit to induce immune serum (polyclonal antibody).ELISA analysis showed that the titer for the polyclonal antibody was 1:128,000.Western Blot analysis showed that the antibody could react specifically with the EPL-1 protein.(3)The expression dynamics of CvBV EPL-1 protein in host Plutella xylostella larvae were analyzed using the EPL-1 polyclonal antibody.The results showed that the expression increases gradually after parasitization.Expression can be detected in plasma 1 day post-parasitization(p.p.)and in hemocytes 3 days p.p.Expression peak appeared at 6 d p.p.both in plasma and hemocytes.(4)The transcription dynamics of CvBV EPL-1 gene in host Plutella xylostella larvae were analyzed using quantitative real-time PCR.The results showed that EPL-1 gene transcripts can be detected in the host larvae as early as 2h p.p.and continued to be detectable for 6 days p.p.There are two transcription peaks in the host(2 d p.p.and 5 d p.p.).But the patterns of transcription dynamics are different in different tissues.In brain,transcription was detected at 2h p.p.and the peak appeared at 12h p.p.In midgut, transcription was detected at 6h p.p.and the peak appeared at 24h p.p.In hemolymph, transcription was detected at 12h p.p.and the peak appeared at 6 days p.p.The results showed that the transcription increases gradually after parasitization.The transcription dynamics of EPL-1 gene corresponds to the expression dynamics of EPL-1 protein.