Optimization Of The In Vitro Culture Conditions Of Haemonchus Contortus Extraction Of H11 And Enzymatic Activity Analysis Of Aminopeptidase Of The Native H11

Haemonchosis was a parasitic disease caused by Haemonchus contortus.It could infected many ruminants including cattle, sheep and so on. The disease mainly used chemicals to cure,but medicine remains and environment pollution were very seriously.It also had drug resistance.To control the disease effectively,it is important in the advanced development of vaccine of Haemonchosis.H11 protein was integrated membrane glucoprotein compound,and molecular weight was 110ku. As hidden antigen, H11could provoke high-level protection of serum antibody. The dada from the exceperiments showed us H11 was most effective irnrnunogen isolated from Haemonchus contortus.The male,female worm burdens and faecal egg eounts could be decreased by70%,80% and 90% respectively after immunization with this antigen. But because of the inability of prokaryotic system on posttranslational processing,modification,glycosylation expression to the product, the immunoprotection of recombination protein was not as good as native antigen.Now the obtain in H11 of adult Haemonchus contortus from natural infection ovine abomasums, cost much and hard to operate. Restriction by mang factors, such as seasonality and gradient of infection of goat itself, frequently mixed other nematode. Because of the impurity and quantity indeterminacy,it cound not satisfy a great quantity demand of scientific research. It is urgent to find a method to obetin a great quantity of Haemonchus contortus using in vitro culture. This study examined the effect and interrelation of different temperatures, pH of the media,and four commercial media on development of H. contortus in vitro culture, in further to improve the in vitro culture conditions of H. contortus. Extracted and purified native Hll of cultivating L4 larvae and adult H.contortus from natural infection, and analyzed enzymatic activity of aminopeptidase. This will lay the foundation to obtain native H11 utilizing in vitro culture and development native antigen vaccine of Haemonchus contortus.1. Under equal status,within a certain Temperature Range,it was direct rate between temperature and hatchability of eggs and breeding rate. The optimal temperature of eggs breeding was 34℃,hatchability reached up to 97%.2. Under equal status,in NCTC135,NCTC109,and M199 culture medium L3 developed to L4 advanced stage,but the larvae in medium F12 could not developed a step further,and stopped at the early fourth stage.More than 99% developed to L4 in NCTC135, among the four. 3. commercial media NCTC135 had the highest survival rate.4. When the pH value was 5.8-6.2,no more than 50% L3 could develop to L4,at PH6.5-6.7,the synchronization of development and the yield of L4 were promoted.The optimum PH value was6.7.5. Extracted and purified native H11 of L4 larvae and adult H.contortus successfully, the result of SDS-PAGE analysis showed that the molecular weight of Hll protein was 110ku. Expression of H11 protien in both L4 larvae and adult H.contortus,and it expressed mainly in adult H.contortus.6. Enzymatic activity analysis of aminopeptidase to L4 larvae and adult H.contortus H11 protien confirmed it to be an Ap, with both ApM and ApA activity.7. Evaluated sensitivity of H11 enzyme inhibitor showed that its activity could inhibite by both EDTA and phenanthrolene.