Identification And Expression Of Ss-ast-1 Astacin Metalloproteinase Encoding Gene Of Strongyloides Stercoralis

The nematode is one of the metazoan animals which have abundant species.Due to its simple structure,some nematodes have been used as the model organisms for neural and evolutionary research,such as Caenorhabditis elegans.However,among the nematodes,there are a class of nematodes that infect animals,also known as parasitic nematodes,which have brought great harm to agricultural and human healthy.According to WHO’s research,about one in eight people are infected with parasitic nematodes,Strongyloides stercoralis is one of those nematodes.The disease caused by S.stercoralis is called strongyloidiasis,one of the widespread but easy neglected tropical disease(NTD)in the world.It is mainly prevalent in areas with poor sanitation such as Africa.At present,there is no simple technique to diagnose this disease,and no vaccine can be used for prevent strongyloidiasis.Astacin metalloproteinase is a metalloproteinase that widely exists in a variety of species.Studies had shown that this proteinase plays an important role in the infection and the growth of nematodes,it is expected to become a new target for detection and vaccines.However,little is known about this molecule in S.stercoralis.This study firstly analyzed the transcriptome of S.stercoralis in various stages and selected the proteins presumed to be astacin metalloproteinases,and then chose the genes to be studied by comparing amino acid sequences with the published sequences of metalloproteinases.Finally,Ss-ast-1 was selected as the target gene.Ss-AST-1 was expressed in E.coli and its protease activity was detected.The expression pattern of Ss-ast-1 in S.stercoralis was investigated by microinjection,and polyclonal antibodies were also produced.(1)Ss-ast-1 was selected as the target geneFrom the transcriptome data of S.stercoralis,49 genes annotated encoding metalloproteinases can be obtained.The amino acid sequences of these metalloproteinases were aligned together with the sequence of astacin metalloproteinases and the phylogenetic tree was constructed for selecting metalloproteinases,and finally Ss-ast-1 was chosen as the target gene.(2)Molecular characterization of Ss-ast-1 DNA sequence and amino acid sequenceThe c DNA sequence of Ss-ast-1 was obtained from Wormbase Parasite.The coding region is 1905 bp and encodes 634 amino acids.According to the analysis results,the first 32 amino acids is signal peptide,and the amino acid sequence comparison revealed that the astacin metalloproteinase Ss-AST-1 has conserved structural domains,such as Zine-binding,Met-turn,and EGF-like domain.Three-dimensional homology modeling analysis of Ss-AST-1 was also conducted.The predicted length of the Ss-ast-1 promoter is 468 bp,and it has many promoter regulatory elements which are conserved in eukaryotes.(3)Transcription level of Ss-ast-1 and localization of Ss-AST-1 in S.stercoralisTranscriptome analysis revealed that Ss-ast-1 was expressed in all developmental stages of S.stercoralis,especially in i L3.Transgenesis in S.stercoralis revealed SsAST-1 was expressed in F1 eggs.(4)Prokaryotic expression of Ss-AST-1Ss-AST-1 was expressed in inclusion body of E.coli and the purified protein was used for enzyme activity assay and for preparing polyclonal antibodies.Western blot analysis showed that polyclonal antibodies can better recognize whole worm proteins,indicating that the protein’s immunogenicity is good.This study investigated the genetic structure,expression patterns and function of Ss-ast-1 in S.stercoralis.It revealed that Ss-ast-1 is expressed in eggs and has a good immunogenicity.The findings provide the theoretical basis of new vaccine development and detect methods.