Identification Of M13 Recombinant Phage And Effect On Chlamydia Trachomatis And Chlamydiadophila Caviae

[Background]In recent years chlamydia trachomatis urogenital infection has been the most common sexually transmitted disease at home and abroad.The incidence of this disease is increasing year by year,and it has become the first sexually transmitted disease.According to WHO statistics,about 92 million new Chlamydia trachomatis infections occur worldwide every year.About two-thirds of Chlamydia trachomatis infections have no clinical symptoms and are easily neglected,leading to persistent infection of Chlamydia in the host,resulting in a series of serious complications and sequelae.It is difficult to eradicate Chlamydia thoroughly,because Chlamydia is a special bacterium.Considering unique characteristics of chlamydia antibiotics treatment may not be effective.Chlamydia phage is a kind of bacteriophage with Chlamydia as its host,which can cause the initial Chlamydia lysis and pass on to the next generation by simple adhesion.At present,Chlamydia phage has been used as a biological preparation for the treatment of Chlamydia infection.Therefore,Chlamydia phage may be an important way to solve this problem,but no Chlamydia trachomatis phage has been found.Previous work in our laboratory showed that Chlamydia trachomatis could be inhibited by purified Chlamydia phage capsid protein Vp1.The analysis of Vp1 protein suggested that IN5 loop was the most important functional region.[Objective]To explore the effect of M13-IN5 recombinant phage on Chlamydia trachomatis E and Chlamydiadophila caviae,.[Content]To detect the titer of M13-IN5 recombinant phage and to study the expression of M13-IN5 recombinant phage IN5 protein.To interfere with CtE by constructed M13-IN5 recombinant phage,and the changes of Chlamydia trachomatis in 36,48,60 and 72 hours were observed by light microscopy.To interfere with Chlamydiadophila caviae by constructed M13-IN5 recombinant phage,and the changes of Chlamydiadophila caviae in 20h,24h,28h,32h hours were observed by immunofluorescence microscope.We observed the location of M13 phage and M13-IN5 recombinant phage by confocal laser scanning microscope in 36 h.[Methods]The titer of recombinant bacteriophage was determined by double-layer plate method.We use DNA Extraction Kit extracting bacteriophage DNA,.Identification was carried by PCR,digestion,the sequencing.Phage protein was extracted and was used to detect the expression of recombinant IN5 cycloprotein by Western blot.The activity of on M13-IN5 recombinant phage were evaluated by double-layer plate method.During the cultivation of CtE,M13 phage and M13-IN5recombinant phage with the titer of 10¹¹ PFU/ml were added to the plate,at the same time,CtE control group and blank control group were set up.The localization of M13phage and M13-IN5 recombinant phage was detected by confocal laser scanning microscope in 36 hours.The M13-IN5 recombinant phage was inoculated with CtE into dense monolayer Hela cells.After 36,48,60 and 72 hours of intervention,the effect of M13-IN5 recombinant phage on CtE was observed by ordinary light microscope.The M13-IN5 recombinant phage was inoculated with Chlamydiadophila caviae into dense monolayer Hela cells.After 20h,24h,28h,32h hours of intervention,the effect of M13-IN5 recombinant phage on Chlamydiadophila caviae was observed by immunofluorescence microscope.[Results]The recombinant M13-IN5 phage was obtained by DNA recombination technique.The phage titer was obtained by counting the plaque.LB plate culture showed that the plaque forming unit of the recombinant phage was3.05 x10¹¹PFU/ml,suggesting that the recombinant phage had biological activity.M13-IN5 recombinant phage group had a strong protein band at about 55 kDa,which was exactly the same size as the fusion protein of IN5 protein and M13 phage pIII coat protein?58.2 kDa?.There was no band at the corresponding position between M13 phage and E.coli ER2738 group,which indicated that the recombinant phage IN5 gene could be expressed normally.Immunofluorescence analysis showed that phage fluorescence was overlapped with Chlamydia inclusion body fluorescence.M13-IN5 recombinant phage showed significant inhibitory effect on Chlamydia trachomatis and Chlamydia guinea pig.The number of inclusion bodies in M13phages and M13-IN5 recombinant phage decreased after CtE infection 36 h,60h and72 h?P<0.05?,and the number of inclusion bodies in M13-IN5 recombinant phage was higher than that in M13 phage after CtE infection 36 h,72h?P<0.05?.But the number of M13-IN5 phage inclusions and M13 phage inclusions were not significantly different between after after CtE infection 48 h,60h?P>0.05?.The number of inclusion bodies of M13 phage and M13-IN5 recombinant phage group decreased after Chlamydiadophila caviae infection 28 h,32h?P<0.05?,and the inclusion bodies'number of M13-IN5 phages group was higher than number of M13phages at Chlamydiadophila caviae infection 28h,32h?P<0.05?.But the number of M13-IN5 recombinant phage inclusions and M13 phage inclusions were not significantly different between at Chlamydiadophila caviae infection 20 h,24h?P>0.05?.[Conclusion]M13-IN5 recombinant phage has bioactivity and can successfully express IN5 protein.In vitro,the recombinant phage can enter the inclusion body of Chlamydia trachomatis and inhibit the infection of Chlamydia trachomatis and Chlamydiadophila caviae.