Activity And Pathogenicity Of L.Interrogans M23 Metalloendoneptidases And Screen And Identification Of Nontypeable H.Influenzae Predonminant Epitopes

Part-Ⅰ Activity and pathogenicity of M23 superfamily metalloendopeptidases of Leptospira interrogansBackground:Leptospirosis is a zoonotic infection disease caused by pathogenic Leptospira genospecies.In china,leptospirosis is one of the nationally-prevented and-controlled major infectious diseases.According to pethogenicity,Leptospira can be classified into pathogenic and non-pathogenic groups.Among pathogenic Leptospira,Leptospira interrogans is the most prevalent genospecies.In particular,L.interrogans serogroup Icterohaemorrhagiae serovar Lai has been confirmed as the predominant serogropu and serovar prevailing in China.Pathogenic Leptospira has a powerful invasiveness that enable the pathogen fast pass through skin and mucosa to invade human body as well as pass through vessel wall to diffuse multiple internal organs and tissues.However,the material basis and mechanism of leptospiral invasiveness remain poorly understood.Metalloprotease(MP)is a large group of enzymes hydrolyzing proteins or polypeptides.In the genome of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai,there are multiple genes which products were annotated as MP,but enzymatic activity and pathogenicity of these gene products have not been characterized yet.Methods:The signal peptide,transmembrane region and functional domains in LA2582 and LA2901 genes of L.interrogans serogroup Icterohaemorrhagiae serovar Lai were analyzed by bioinformatic softwares.The prokaryotic expression systems of the extracellular regions of LA2582 and LA2901 genes were generated.The target recombinant expression products,rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad gel image analysis system were used to detect the effects of rLA2582 and rLA2901 expression and extraction.The purified rLA2582 and rLA2901 were intradermally injected into rabbits for immunization to obtain their antisera and IgGs.The activity of rLA2582 and rLA2901 hydrolyzing fluorescence-labeling Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans pentapeptide substrate was detected by fluorospectrophotometry,and then the Km and Kcat values were determined.SDS-PAGE was used to detect the activities of rLA2582 and rLA2901 hydrolyzing collagen type-I(COL1)and fibronectin(FN),the common extracellular matrix(ECM)molecules.Spectrophotometry was applied to detect the activity of rLA2582 and rLA2901 hydrolyzing Congo red-labeling elastin(ELN).Using real-time fluorescent quantitative RT-PCR and Western Blot assay,the mRNA,protein expression and secretion levels of LA2582 and LA2901 genes of L.interrogans strain Lai during infection of HUVEC were detected.Results:The bioinformatic analysis showed that both the LA2582 and LA2901 gene products were predicted as Zn2+-dependent Gly-Gly metalloendopeptidases belonging to M23 superfamily with a signal peptide,a transmembrane region and M23 motif HXH.The generated prokaryotic expression systems of LA2582 and LA2901 genes effectively expressed the target recombinant proteins rLA2582 and rLA2901.Both rLA2582 and rLA2901 could hydrolyze the pentapeptide substrate Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans with the Km or Kcat values of 126.543 μmol·L-1 or 4.675 s-1 and 190.25 μmol·L-1 or 4.859 s-1.rLA2582 and rLA2901 also could hydrolyze the ECM molecules COL1,FN and ELN.After infection of HUVEC with L.interrogans strain Lai,the mRNA,protein expression and secretion levels of LA2582 and LA2901 genes were significantly increased(p<0.05).Conclusion:The products of LA2582 and LA2901 genes of L.interrogans strain Lai are the Zn2+-dependent M23 metalloendopeptidases with hydrolytic activities of multiple ECM molecules that are closely involved in leptospiral invasiveness.Part-Ⅱ Screening and identification of predominant antigenic epitopes in OMP6 of nontypeable Haemophilus influenzaeBackground:Infection of Haemophilus influenzae causes many diseases in children such as pneumonia,meningitis and tympanitis.According to the presence of capsule and capsular polysaccharide antigens,H.influenzae can be classified into typeable Haemophilus influenzae(THi)using serologically assays and serologically nontypeable Haemophilus influenzae(NTHi).Inoculation of multiple-valence H.influee capsular polysaccharide vaccine can efficiently prevent the infection of THi,but this vaccine has no immunoprotection against the infection of NTHi.Therefore,screening and identification of T and B cell(T-B)-combined antigenic epitopes in outer membrane proteins(OMP)of NTHi will provide a solid basis for further developing antigenic peptide vaccine against NTHi.Methods:The entire OMP6-encoding genes of NTHi isolates were amplified by PCR and the amplification products were sequenced after T-A cloning.By using bioinformatic softwares,the sequence conservation and membrane location of OMP6 were analyzed as well as the T-B-combined antigenic epitopes in OMP6 sequence were predicted.The immunogenicity and immunoreactivity of T-B-combined antigenic epitope peptides displayed by recombinant phage PⅢ proteins(rPⅢ)were determined by Western Blot assay and ELISA.Results:The PCR showed that all the 35 NTHi isolates tested were detectable for OMP6-encoding genes.The identities of nucleotide and amino acid sequences of OMP6-encoding genes from 28 strains in the NTHi isolates were 98.3%-100%and 99.3%-100%,respectively.The OMP6 of NTHi was predicted as an outer membrane superficial protein that contains OMP6-2-25,OMP6-61-86 and OMP6-98-126 predicted T-B-combined antigenic epitopes with high scores.The Western Blot assay and ELISA confirmed that the OMP6-2-25 presented stronger hybridization band with NTHi antisera while 96.9%(59/62),69.4%(43/62)and 74.2%(46/62)of serum samples from NTHi-infected children were positive for OMP6-2-25,OMP6-61-86 and OMP6-98-126 T-B-combined antigenic epitope peptides,respectively.Conclusion:The OMP6-encoding gene is extensively distributed in NTHi isolates with high conserved sequences.OMP6-2-25 is the predominant T-B-combined antigenic epitopes which can be used as the candidate for developing multiple antigenic peptide vaccine against NTHi.