Cloning And Functional Study Of Hc-CAP-15 And Hc-CAP-17 Encoding Genes Of Haemonchus Contortus

Haemonchus contortus is a blood-sucking parasitic nematode of ruminants with a global distribution.Currently,it causes one of the most severe gastrointestinal parasitic diseases in ruminants.The prevention and control of this disease mainly relies on the use of anthelmintics,but long-term and unreasonable excessive use of anthelmintics has led to the emergence of single or even multi-drug resistant strains of H.contortus.The epidemic of haemonchosis has severely restricted the development of animal husbandry.Therefore,it is urgent to seek suitable vaccine candidate molecules and develop commercial vaccines for the prevention and control of haemonchosis.CAP(Cysteine-rich secretory proteins,Antigen 5 and Pathogenesis-related 1,CAP)protein is widespread in prokaryotes and eukaryotes,and its protein types are diverse and functions are complex.Recently,based on the genomics and transcriptomics analysis of H.contortus,it has been shown that there are a large number of CAP protein molecules in this parasite,but its exact function has not yet been resolved.Therefore,this study starts with the functional study of whether the CAP protein can regulate the growth and development of H.contortus,and selects Hc-CAP-15 and Hc-CAP-17 from the reported CAP molecules of H.contortus as the research objects,using transcriptional level analysis,immunohistochemistry and RNAi,preliminary exploration of the regulatory role of these molecules in the growth and development of parasites.(1)Amplification of Hc-CAP-15 and Hc-CAP-17 encoding sequence and protein sequence analysisBased on the genome data of H.contortus,the encoding sequences of Hc-cap-15 and Hc-cap-17 were obtained by designing and synthesizing specific primers.Subsequent amino acid sequence alignment and phylogenetic tree analysis showed that both Hc-CAP-15 and Hc-CAP-17 have a CAP domain composed of four CAP motifs and conserved cysteine residues,which shows that both are typical single domain CAP proteins.(2)Transcriptional level analysis of Hc-cap-15 and Hc-cap-17Fluorescence quantitative real-time PCR was used to determine the transcriptional levels of Hc-cap-15 and Hc-cap-17 in eight developmental stages/sexes of H.contortus,including eggs,first-stage larvae,second-stage larvae,third-stage larvae,fourth-stage larvae,adult females and adult males.Both genes were transcribed in eight stages with the highest level in the fourth-stage larvae for Hc-cap-15,and the second-stage larvae for Hccap-17(3)Localization of Hc-CAP-15 and Hc-CAP-17 in adult H.contortusRecombinant Hc-CAP-15 and Hc-CAP-17 were successfully expressed,purified and then used to immunize New Zealand white rabbits to produce polyclonal antibody.The obtained polyclonal antibodies were purified and then used to localize the expression of these two proteins in the adult H.contortus.Hc-CAP-15 was mainly expressed in the intestinal microvilli,ovaries and epidermis of female adults and in the intestines of male worms,and Hc-CAP-17 was mainly expressed in the intestinal wall and ovary.(4)RNAi of Hc-cap-15 and Hc-cap-17SiRNA was used to knock-down Hc-cap-15,which decreased the transcriptional level of Hc-cap-15.The down-regulation of Hc-cap-15 had no significant effect on the developmental rate of the worms from the third-stage larvae to the fourth-stage larvae,but increased the body length of siRNA treated worms,indicating that Hc-cap-15 has a negative regulatory effect on the growth of the worm.However,neither dsRNA nor siRNA can down-regulate the transcription of Hc-cap-17.(5)Determination of reactogenicity of Hc-CAP-15 and Hc-CAP-17The negative serum of the newborn goats and the positive serum of H.contortus infected goats were used to determine the reactogenicity of the two molecules.Both HcCAP-15 and Hc-CAP-17 can be recognized by the positive serum,suggesting they have good reactogenicity.In summary,this study investigated the expression pattern of Hc-CAP-15 and HcCAP-17 and assessed their importance in regulating the growth and development of H.contortus.The findings lay foundation for future functional and mechanistic studies on these molecules,which will help the understanding of biology of parasitic nematodes.