Preparation And Characterization Of Monoclonal Antibody Against The Native H11 Protein Of Haemonchus Contortus

Haemonchosis is one of the most severe gastrointestinal parasitic diseases in ruminants.The disease has a global distribution and is widely prevalent at home and abroad.It can cause anemia and weight loss in host animals such as cattle and sheep.Acute infection often causes animal death and serious harm.At present,the prevention and treatment of the disease mainly relies on anthelmintic drugs,but the unreasonable use of drugs has made the problem of drug resistance more and more serious,and drug-resistant strains of Haemonchus contortus have appeared all over the world.Therefore,there is an urgent need to develop new control methods to control haemonchosis.Vaccines have attracted widespread attention as a long-term and effective prevention and control strategy.Studies have shown that the natural H11 antigen can induce a high level of immune protection in the host and is one of the most potential vaccine candidates.However,the recombinant H11 antigen obtained in expression systems has poor immune protection,which has hindered vaccine development.The main reason is that the mechanism of natural H11-induced immune protection is not yet clear,but existing studies suggest that the protection effect induced by H11 is mediated by antibodies.Therefore,this thesis took the natural H11 protein as the research object,by producing and characterizing a monoclonal antibody against the natural H11 protein of H.contortus,to provide a basis for further identifying the epitopes in H11 and studying the protective mechanism of H11,which has significant implications for the vaccine development.The specific research contents are as follows:（1）Extraction of native H11 proteinH.contortus adults were collected from the goat abomasum infected with H.contortus,and the H11 protein was preliminarily extracted from the adult homogenate by Thesit,and the purified H11 protein was further extracted by concanavalinum A（Con A）.（2）Production and evaluation of anti-H11 monoclonal antibodyBALB/c mice were immunized with H11 protein.Two monoclonal antibodies（A1E3and A10E1）were produced using hybridoma cell technology.The obtained two anti-H11mAbs were examined by Western-blot,and both could react with H11.The chromosome count of hybridoma cells showed that the two hybridoma cells were stable at the chromosome level.The results of antibody subtype identification showed that the heavy chain of the two monoclonal antibodies was IgG1,and the light chain wasλ.The two mAbs were found to recognize the same antigenic determinant by additive reaction.（3）Purification of anti-H11 monoclonal antibodyBased the evaluation results of the two monoclonal antibodies and the growth status of the cell lines,the hybridoma cell line A1E3 with better stability was selected,and injected into the abdominal cavity of mice to prepare ascites.After purification of the ascites,the antibody titer was measured as 100×2⁹,and the concentration was 1.79 mg/mL.（4）Tissue localization of mAb A1E3 in adult H.contortusThe adult H.contortus was made into tissue sections,and the purified mAb A1E3 was used as the primary antibody for tissue localization.At the same time,the serum of unimmunized mice was used as a negative control.The results showed that the mAb A1E3can specifically bind to the intestinal microvilli of H.contortus adults.（5）The effect of mAb A1E3 on the growth and development of H.contortus larvaeThe purified mAb A1E3 was added to the culture medium of the exsheathed third-stage larvae（x L3）at doses of 5μg/mL,10μg/mL,20μg/mL,50μg/mL,and 100μg/mL,and the IgG1 was used as the isotype control.After culturing for 7 days at 39℃ and 20%CO₂,the developmental rate of larvae from x L3 to the fourth stage larvae（L4）was counted,and the body length and width of the larvae were recorded and analyzed statistically.The results showed that the addition of monoclonal antibody had no obvious inhibitory effect on the development rate and body width of larvae,but when the antibody dose reached 50μg/mL,the monoclonal antibody significantly inhibited the growth of larval body length.（6）The effect of mAb A1E3 on the intestinal aminopeptidase activity of H.contortusThe purified mAb A1E3 was co-incubated with the intestinal homogenate of H.contortus,and then the aminopeptidase activity was detected.The results showed that compared with IgG1,the mAb A1E3 significantly inhibited the aminopeptidase activity.