Cloning,Expression And Characters Of Aspartyl Protease Inhibitor From Haemonchus Contortus

Haemonchus contortus is one of the most important gastrointestinal parasitic nematodes of small ruminants worldwide.This nematode feeds on blood and often causes anemia in the host,even death in lambs.Aspartyl protease inhibitor(API)is one of the important components of the parasite,which associate with the growth,development and invasion,even play roles in evasion and regulation the immune response of the host.It has been suggested that protease inhibitors are involved in the parasite-host interations and are critical for parasite survival.In this research,the gene encoding API was cloned,expressed,and partial of the biological characters of the recombinant protein were studied.1.Cloning and expression of recombinant API from H.contortusUsing a pair of special primers based on the API sequence of H.contortus,a DNA fragment of 681 bp was produced by RT-PCR.The fragment was cloned into pMD18-T vector,sequenced and analyzed.It was found that this gene was 94%similar with the gene of H.contortus API.Then the prokaryotic expression plasmid pET32/API was constructed by subcloning the ORF of API into the vector pET-32a(+).The recombinants were transformed into BL21 and induced by isopropyl-B-D-thiogalactopyranoside(IPTG).SDS-PAGE analysis showed that the recombinant protein was expressed with the molecular weight of 48×10.The recombinant protein was analyzed by Western blot.The result showed that the recombinant protein could be recognized by the serum from the goat infected with H.contortus.2.Enzyme inhibition assay of the recombinant APIThe recombinant API was thoroughly mixed with equal mass of pepsin or trypsin at 37? for 30 minutes,followed by adding the hemoglobin as substrate and incubating at 37? for 30 minutes.The resulted products were analyzed by 12%SDS-PAGE.The inhibition efficiency and optimal reactive conditions for API were also checked by photocolorimetric method.SDS-PAGE results suggest that the recombinant API can inhibit the activity of pepsin significantly,but can't inhibit that of trypsin.It was found that enzymatic activity of pspsin was 63%decreased by adding the API.Resuts suggest that recombinant API is a heat-resistant inhibitor,with the optimal temperature from 37? to 50?.3.Differential expression of API in different life stages of H.contortusThe total RNA was extracted from eggs,L3,xL3,female and male adults and reverse transcribed into cDNA.The primers for quantitative PCR were designed based on the sequences of H.contortus API and ?-Tubulin.After verifying the efficiency of the amplifications,the optimal primers were choosen.The real time PCR results showed that the lowest expression of API was in female adults,while the highest expression was in the L3s,with about 40 times of the female adults.4.The localization of API in H.contortusThe frozen sections of adult worms were detected by the method of immunofluorescence technology with anti-API serum as the first antibody in order to analyze the localization of API in H.contortus.The result suggested that API was expressed in both female and male adults,which located in the intestinal tracts and subcutaneous tissue.However,the expressions of API in females were lower than that in males.5.Effects on cytokine expression in the goat PBMCs induced by the recombinant APIGoat PBMCs were incubated with the recombinant API(0,10,20 and 40 ?g/mL)and the transcription levels of different cytokines(IL-2,IL-4,IL-10,IL-17,IFN-? and TGF-p)were quantitied by real-time PCR using the method of 2-??Ct.Results showed that the mRNA transcription levels of IFN-?,IL-4 and IL-10 arised significantly,while those of IL-2,IL-17 and TGF-? were not significant different.