Study On Resistance Related Protein In Haemonchus Contortus With Ivermectin Resistance

Parasitic nematodes are worldwide spread,especially the Haemonchus contortus,which is one of the most important parasitic nematodes that harm ruminants.Although the anthelmintics can better control nematode infections,the anthelmintic-resistance of the worms can not be ignored any more,and it has become a hot issue of public concern.Being shortage of studying methods,there is still lack of information on the current status and mechanism of anthelmintic-resistance.In view of these problems,this study intends to take ivermectin-resistant Haemonchus contortus as the research object,and on the basis of the full release of protein from the broken body and eggs,Shot-gun LC MS/MS analysis was applied to the proteins to discover proteins related to drug resistance,the detailed results were as follows:(1)Screening of egg cracking method of H.contortusFirstly,clean eggs were obtained by the floatation method of sugar and salt water,and then were broken by enzymolysis method,repeated freeze-thaw method,strong acid and alkaline solution method,and combination of hydrolysis and repeated freeze-thaw methods,respectively.Later,the number of unly sed eggs were counted and the protein were verified by SDS-polyacrylamide gel electrophoresis.It was finally determined that when 2 mL of chitinase solution was added to 1 mL of worm egg suspension and lasted for 45 minutes,followed by being placed in liquid nitrogen and frozen for 30 min.After being taken out,it was placed in a 50? water bath for 6 hours.All steps were repeated 3 times.The egg cracking effect was the best,and the cracking rate of the eggs was 93%.(2)Screening of the cracking methods of the H.contortus larvaeFor the third stage larvae of Haemonchus contortus,liquid nitrogen grinding method,repeated freeze-thaw method,liquid nitrogen grinding method and repeated freeze-thaw method with de-sheathing larvae were used.The results showed that liquid nitrogen grinding method was the effective method for larvae breaking.The lysis of the worm was obvious,and the cracking rate of the worm was 100%.(3)Shotgun LC MS/MS analysis of eggs and larval proteins of H.contortusThe Shotgun LC MS/MS analysis was carried out on the eggs and larval proteins of H.contortus,a total of 528 proteins were finally identified in the Uniprot protein database,of which 16 unique proteins were identified in the resistant larvae of H.contortus,2 unique proteins in sensitive larvae,192 unique proteins in sensitive eggs,and 30 common proteins in sensitive eggs,sensitive larvae and drug-resistant larvae.These proteins were mainly involved in proteolysis,metabolism,transcription,ion transport,electron transport,molecular binding,protein folding,signal transduction and so on.At the same time,four proteins related to the resistance of worms-the peptidase C2 domain,cathepsin L cysteine protease,superoxide dismutase[Cu-Zn]and actin were found in the proteins.(4)Western-blot test of the protein from H.contortusAccording to Western-blot results,the size of the protein from H.contortus was in 14-120 KDa,which confirmed that there were differences in the protein of different strains and stages of the worm,and some of the protein could be recognized by sheep positive serum.The identification of the proteins mentioned above provided important information for the research on the development,infection,immune escape,screening of vaccine antigens and drug targets,and anthelmintic-resistance mechanisms of H.contortus,and it also provided basic information for understanding the molecular background and revealing the mechanism of anthelmintic-resistance.