Prokaryotic Expression And In Vitro Activity Of Ancylostoma Ceylanicum Platelet Inhibitor

Ancylostoma ceylanicum is an animal-derived hookworm that can develop into an adult in the intestinal tract of human and pet(dogs and cats),causing humans iron deficiency anemia and malnutrition.Hookworm platelet inhibitor(HPI)is a secreted protein of hookworm with strong pathogenicity,which can inhibit platelet aggregation and adhesion in human intestine and facilitate blood sucking.Meanwhile,it also can be used as a candidate molecule for hookworm vaccine.In this paper,cloning and expression of Ancylostoma ceylanicum platelet inhibitor(Ace-HPI)gene and its activity in vitro were studied,which were of great significance for the prevention and treatment of this disease.1.Cloning and sequence analysis of Ace-HPI gene.Referring to the nucleotide sequence of Ac-HPI gene(Gen Bank: AF399709.1),specific primers were designed to amplify the Ace-HPI gene using Ancylostoma ceylanicum c DNA as a template.The transcription level of Ace-hpi m RNA in the L3 larval and adult stages was detected by fluorescence quantitative PCR,and its amino acid sequence was bioinformatically analyzed using online software.The results showed that the full length of Ace-HPI gene was 603 bp,encoding 200 amino acids,and had 91% homology with amino acid sequence of Ac-HPI.The expression level of Ace-hpi m RNA in adult was the highest(p<0.01),followed by L3 larvae stimulated by serum.The protein has a signal peptide sequence of 17 amino acids,its theoretical molecular mass and isoelectric point is 22.5 k Da and 7.0,respectively,and it is a hydrophobic protein without transmembrane domain.Besides,there is six alpha helices and six beta folds in its tertiary structure.2.Prokaryotic expression of Ace-HPI gene.The amplification product of Ace-HPI gene(without signal peptide sequence)was connected to p ET-28 a vector and transferred into the competent cell E.coli Rosetta-gami2(DE3).After double enzyme digestion and sequencing verification,the expression was induced by isopropyl-penta-d-thiogalactoside(IPTG).The results showed that the recombinant protein was mainly expressed in soluble form,and the optimal expression condition was induced for 24 h at 28℃ with 0.5 m M IPTG.The purified target protein could be obtained by Ni column affinity chromatography.3.Tissue localization of Ace-HPI in adult Ancylostoma ceylanicum.The anti-serum was prepared by immunizing Kunming mice with the purified recombinant protein Ace-HPI.The antibody titer was detected by ELISA.The adults of Ancylostoma ceylanicum were fixed,embedded in paraffin and sliced.The adult slices were reacted with mouse anti-Ace-HPI serum as the first antibody and the FITC-conjugated goat anti-mouse Ig G antibody as the second antibody,and observed under fluorescence microscope.The results showed that the serum titer of anti-Ace-HPI was as high as 1:409600,and the Ace-HPI was mainly localized in the esophagus and cephalic gland of adult Ancylostoma ceylanicum.4.Detection of Ace-HPI activity in vitro.Anticoagulant blood was collected from dog veins and platelets were isolated by gradient centrifugation.The purified Ace-HPI recombinant protein was incubated with platelets,and its activity in vitro was detected using microplate and spectrophotometer.The results showed that the recombinant protein inhibited platelet aggregation,but the inhibition of platelet adhesion was weak.5.Effects of Ace-HPI on PBMCs proliferation and cytokine expression.Peripheral blood mononuclear cells(PBMCs)were isolated from the peripheral blood of healthy dogs and stimulated in vitro with different concentrations of Ace-HPI recombinant protein.The proliferation of the PBMCs were detected by CCK-8,and the total RNA of the PBMCs was transcribed into the first strand c DNA,and the transcriptional level of cytokines was detected by fluorescence quantitative PCR.The results showed that Ace-HPI could stimulate the proliferation of the peripheral blood lymphocytes and the secretion of IL-10,IL-13 and other cytokines.The above-mentioned results indicated that Ace-HPI,a member of the CAP superfamily,was highly expressed in E.coli Rosetta gami2(DE3).It had a high expression level in adults and located in the cephalic gland and esophagus.It has the function of inhibiting platelet aggregation,and can stimulate the proliferation of peripheral blood lymphocytes and high expression of cytokines IL-10 and IL-13.These findings lay a foundation for further research on the biological function of the protein and its potential as a candidate molecule for hookworm vaccine.