Study On The Sequence Characteristics Of ApxIVA Gene And The Immunization Effects Of RApxIVA/rTbpB Subunit Bacterine In Mice

Actinobacillus pleuropneumoniae(APP) is the causative agent of porcine infectious pleuropneumonia syndrome(PIPS),a fibrinous,exudative,hemorrhagic,necrotizing pleuropneumonia affecting all ages of pigs.The virulence of A.pleuropneumoniae is known to be variable.BiotypeⅠhas been divided into 13 serotypes and biotypeⅡinto 2 serotypes, for a total of 15 serotypes.And virulence factors to compose the virulence of A.pleuropneumoniae are complex.Furthermore,strains of different serotype contain different virulence factors.Though widely used,inactive vaccine could not induce mice to absolutely resist attack of APP for reasons mentioned above.The object of this study is to find the effects of two proteins,transferin binding protein B (TbpB) and actinobacillus pleuropneumoniae RTX toxin IVA(ApxIVA) on the protection ability of inactive vaccine in mice.At first part of the study,the characteristics of apxIVA was analysed by bioinformatics method to find the conserved region and variable region.For the variable region,repeat sequence analysis was conducted to confirm the region of repeat region,the repeat units and the arrangement of them.For the conserved region,fragments encoding two characteristic amino acid sequence,N end hydrophobic region and middle part potential acylation sites of ApxIVA,were PCR amplified to construct expression plasmids apxIVA1-pET32a and apxIVA1-pET32a.Another expression plasmid tbpB-pET32 constructed in previous research and expression plasmids constructed in this study were transformed into expression host cell E.coli BL21 to express rApxIVA1,rApxIVA2 and rTbpB with the induction of 0.6 mmol/L IPTG for 3 hours.Results of western-blot analysis showed that rApxIVA1 and rTbpB could strongly bind antibody react with positive sera prepared by inoculating mice with live bacteria.On the other hand,rApxIVA2 only possesses weak antibody reaction ability.25μg of rApxIVA1 and 25μg of rTbpB were used as subunit vaccines to immunize mice for three times with interval of two weeks by intraperitoneal injection.Results of antibody detection indicated that two recombinant proteins could induce mice to produce high level of antibodies against them after two times of immunization.Inactive vaccine prepared by inactivating the culture of strain 4074 by formalin was used to immunize mice with dosage of 1×10~9CFU per mouse using the same method as used for subunit vaccines.Results of antibody detection indicated that inactive vaccine could induce mice to produce antibodies against ApxⅠ,ApxⅡand LPS1.The level of antibodies against CPS1 was not significant with those in negative control.The level of antibodies against ApxⅠ,ApxⅡand LPS1 were all significantly lower than those produced by infection of live bacteria(P＜0.01).When the mice were vaccinated with vaccines prepared by mixing recombinant proteins with formalin-inactivated bacteria,the level of antibodies against ApxⅠand LPS1 were significantly lower than those induced by vaccination with single inactive vaccine.But the antibodies against recombinant proteins were not affected by the addition of bacteria.One week after the third immune,spleens of three mice in each group were collected for proliferation activity test.Results of MTT assay demonstrated that the proliferation activity of spleen cells from mice vaccinated with 50μg recombinant protein(25μg of rTbpB and 25μg rApxIVA1) is significantly stronger than other groups.The proliferation activity of spleen cells from mice vaccinated with inactive vaccine is significantly weaker than other groups.Part of the mice,30%in group vaccinated with rTbpB,20%in group immunized by rApxIVA1,40%in group vaccinated with the mixture of both rTbpB and rApxIVA1 survived from the challenge of strain 4074,serotypel,from which the nucleotides for the construction of tbpB-pET and apxIVA1-pET derived.Compared to single inactive vaccine,the protection rate against challenge of strain 4074 was raised by immunization with the mixture of inactive vaccine and either rTbpB,rApxIVA1 or both.After challenge of strain 4074,only 50%of mice survived in groups vaccinated with single inactive vaccine.When combined with rThpB or rApxIVA1,the protection rates were improved to 60%.Further more,80%mice in group vaccinated with the mixture of inactive vaccine and both recombinant proteins.The number of APP either in lungs of mice died right after challenge or survived for one week from challenge day were detected by Real time PCR method.Compared to the number of APP in lungs of dead mice,there was significant difference among groups vaccinated with different vaccines.Vaccination with rTbpB helps to decrease the number of APP in lungs.No significant change is detected in groups vaccinated with inactivate vaccine.The number of APP existed in lungs was raised greatly,almost ten times of those detected in dead mice. After challenge with strain Fem,serotype 6,almost all the mice tested died,with the exception of only mouse in group vaccinated with the mixture of inactive vaccine and two recombinant proteins.The results in challenge test demonstrated that,when used as vaccine, two recombinant proteins could provide some degree of protection only to the challenge of same strain.The significant characteristic,which was discovered during the course of clone,of apxIVA gene is that repeat units in repeat region would lose and finally change into non-repeated sequence when they duplicate outside genome of APP.Sequence analysis shows that apxIVA gene consists of 4 kind of repeated units(with length of 159bp,144 bp,135 bp and 84 bp,respectively).The arrangement of repeat units in apxIVA gene of strain L20,strain HV114 and strain 4074 can be shown as[(P144 bp)(P159 bp)(P135 bp)]m[(P144 bp)(P159 bp)(P84 bp)(P144 bp)(P159 bp)(P135 bp)]n(P144 bp)(m=0 or 1,n≥1).Primers were selected from sequences just adjacent the repeat structure to amplify this repeated area.Gel electrophoretic analysis of PCR products showed there were more than one bands.Bands with size of about 1800 bp,1200 bp,600 bp from the PCR products amplified from genome of 4074 were reclaimed and transformed into E coli after being ligated with pGMT vector. Sequencings results demonstrated that obtained plasmids from the clones of different size of fragments contained completely same inserted nucleotide sequences,which was part of amplification area sequence,39 bp from the binding site of upstream primer to repeat region, 36 bp from the binding site of lower primer to the 3end of repeat region,the other 144 bp from one repeat of 144 bp repeat unit located at 3end part of repeat region.T clone results indicated that PCR amplification from the same template gave different size of products.And they would get into same sequence with the loss of all repeated sequence after replication in plasmid.Whether this phenomenon has causal association with the self-processing "clip and link" activity observed by Osicka,or whether the same phenomenon occurs in bacterial genome replication need further study to discover.