Biological Characters Of Heat Shock Protein 70 And Catalase From Haemonchus Contortus

Haemonchus contortus is one of most import,ant parasites of ruminants,which causes huge economic losses in farm industry.With the progresses of-omics in this nematode,finding novel measures for prevention and controlling of this parasitebecomes possible.Previous researcher in our lab found that the heat shock protein 70(Hsp70)were up-regulated during the transit from the third stage larval(L3)to active L3(aL3),while the catalase was down-regulated.In order to explore the roles of these differentially expresed proteins in parasite infection,partial biological characters were studied in this paper.1.Gene Clone,prokaryotic expression and characters analysis of Hsp70 from H.contortusThe Hsp70gene sized as 1941 bp was amplified by RT-PCR.The fragment was cloned into pMD19-T vector and verified by sequence analysis.The ORF of Hsp70 wassub-cloned into vector pET-28a(+).Then the recombinant prokaryotic expressing vector pET28a-Hsp70 was transformed into Escherichia coli BL21,following by inducing with IPTGSDS-PAGE showed that the recombinant protein was expressed with the size of 75kD.Result of Western blot found that the recombinant protein could be recognized by the serum from goat infected withH.contortus.The recombinant Hsp70 could bind to PBMCs from goats in vitro by immunofluorescence antibody technique.The secretion of NO bygoat PBMCs were increased after incubation with the recombinant Hsp70.Histochemistry assay was used to study the localization of Hsp70 in adult worms.The results showed that Hsp70 were expressed in the intestinal tracts,muscles and the subcutis.2.Gene Clone,prokaryotic expression and characters analysis of catalase from H.contortusThe gene encoding H.contortuscatalase was amplified by RT-PCR.The PCR products were purified and ligated with pMD19-T vector.After identification with enzyme digestion and sequence analysis,the ORF of catalase was cloned into prokaryotic expression vector pET-28a(+).The recombinants were transformed into E.coli BL21,following by inducing with IPTGSDS-PAGE showed that the recombinant protein was expressed with the size of 60kD.The recombinant proteinshowed the catalytic activities.Result of Western blot found that the recombinant catalasecould be recognized by the serum from goat infected withH.contortus.Immunofluorescence assayshowed that the recombinant catalase could bind to goatPBMCs in vitro.After incubation goat PBMCs with the recombinant catalase,the secretion of NO increased significantly.Results of histochemistry assay showed that the catalase was expressed in the intestinal tract,muscle and the subcutis of the adult worms.3.Knockdown of Hsp70 in aL3 by RNA interferenceThree pairs of siRNA(S1-S3)and a pair of no sense RNA were designed based on Hsp70 gene.H.contortusaL3 were soaked with siRNA for 3 days.The expression of Hsp70 was tested by real time PCR.Results showed that each pair of siRNA had suppressed the transcriptions of Hsp70.This paves the way for loss-function studies in the future.