Immunoprotective Effects Of H11 And CAP Proteins Of Haemonchus Contortus

Haemonchus contortus is a highly pathogenic hemorrhagic parasite on the gastric ruminants of small ruminants,which can cause haemonchosis.Major clinical symptoms include anemia,malnutrition,progressive wasting,diarrhea and even death.The epidemiological investigation showed that the haemonchosis was distributed worldwide and caused great economic loss to the breeding industry.At present,drug prevention and control is still the main method for the disease.However,the long-term overuse of anti-worm drugs has caused the emergence of drug resistance in the worm and the residue of animal products and environmental drugs.Under such circumstances,the research and development of effective anti-worm vaccine against the disease is of great importance.Natural H11 antigens provide adequate immune protection,but recombinant forms expressed in expression systems such as Escherichia coli,yeast,insect cells and Caenorhabditis elegans are far less effective.Studies have shown that H11 is an intact membrane glycoprotein complex with a high degree of glycosylation,and the immune protection of H11 is closely related to the glycosylation modification.The expression system of CHO cells has perfect post-translational modification,especially glycosylation modification.Therefore,H11 protein is very suitable for protein expression in CHO expression system.In addition,the CAP protein superfamily has caused increasing attention in recent years,and various studies have shown that it has a strong immune protection against nematode infection.In this study,lg K and GL were selected as signal peptides for protein expression.Firstly,Hc-H11-2 and Hc-cap-35 genes were expressed in adherent CHO cells,and then transferred to the expression of proteins in serum-free suspended CHO cells.After that,the prokaryotic expression of Hc-cap-35,Hc-cap-37 and Hc-cap-45 genes were carried out,and finally the recombinant proteins were immunized in animals to study their immune protective effects.(1)Secretion expression detection of signal peptide and protein expression of Hc-H11-2 and Hc-cap-35 genes in adherent CHO cellsAccording to relevant research reports,lgK and GL were selected as signal peptides for protein expression,adding His-tag was conducive to protein purification,and pc DNA3.1(+)eukaryotic expression vector was constructed to express Hc-H11-2 and Hc-cap-35 genes in adherent-CHO cells.Western blot results showed that both target genes were successfully expressed,and lg K signal peptide significantly promoted the secretory expression of target protein.(2)Protein expression of Hc-H11-2 and Hc-cap-35 genes in suspended CHO cellsDue to transient transfection,CHO adherent cell culture could not produce enough protein for animal experiments,so it was transferred to the study of protein expression in suspended CHO cells.The expression vector with lg K signal peptide was selected for transfection,and about 3mg of Hc-H11-2 protein could be successfully purified from the medium,but not enough Hc-cap-35 protein was obtained for animal experiments.(3)Prokaryotic expression of Hc-cap-35,Hc-cap-37 and Hc-cap-45 genesAccording to reports,all 3 genes had signal peptides.After removing the signal peptides,adding His-tag to construct the vector of p E-sumo for prokaryotic expression,about 2mg could be purified from the supernatant for subsequent animal experiments.(4)To study the immune protection of eukaryotic and prokaryotic recombinant proteinsCHO-H11-2,sumo-CAP-35,sumo-CAP-37 and sumo-CAP-45 recombinant proteins were used for immune test.The immune CHO-H11-2 protein was used for 200μg,and the combination of the immune sumo-CAP-35,sumo-CAP-37 and sumoCAP-45 protein was used for 300μg,Quil A immune adjuvant,subcutaneous injection,three times of immunization and two weeks interval of immunization.The immune adjuvant group was used as the control group and 5000 i L3 larvae were infected.The goats were euthanized 35 days after infection,and the adult count was analyzed by taking the abomasum.In CHO-H11-2 immune group,the results showed that the cumulative mean faecal worm egg counts and worm burdens were reduced by 67.1% and 63.5%,respectively.In the sumo-CAP-35,sumo-CAP-37 and sumo-CAP-45 protein immune groups,the results showed that the cumulative mean faecal worm egg counts and worm burdens were reduced by 41.3% and 70.1%,respectively.All of them have achieved good immune protection effect.In this study,CHO-H11-2 recombinant protein was obtained from suspended CHO cell protein expression system for the first time,and sumo-CAP-35,sumo-CAP-37 and sumo-CAP-45 recombinant proteins were cloned for animal experiments for the first time.Both of the two kinds of recombinant proteins have achieved good immune protection effect,and CHO-H11-2 recombinant protein had better immune effect,which may be related to the glycosylation modification of the protein.These results provide valuable information for the further development of Haemonchus contortus vaccine.