Establishment Of Plant Expression Methods Of Aminopeptidase And Evaluation Of Immune Effect Of Recombinant Protein In Haemonchus Contortus

Haemonchus contortus is an important gastrointestinal parasitic nematode in ruminants,such as cattle and sheep.Adults mainly parasitize in the abomasum of the host,and intake blood from host as its nutrition source,causing anemia and anemia syndrome,or even death.The prevention and control stratiges of haenonchosis mainly rely on the use of chemical drugs,however,long-term and unreasonal usage of chemical drugs lead to the development of anthelmintic resistance,thus the development of vaccine is urgent.Although various expression systems have been applied in the study of vaccine for H.contortus,the effect is limited.Recently,there are increasing reports on the expression of parasite antigens in plants,the use of plant expression systems for the expression of H.contortus antigens can provide a new way for the developmentof vaccines in H.contortus.During the expression of Hc-ap-10 and Hc-ap-8 using the Vector of p RI101 to transient expression the proteins in tobacco leaves,the expression level of codon-optimized Hc-ap-10 was significantly higher that of Hc-ap-10 and Hc-ap-8 without codon optimization.Therefore,it is possible that the optimization of codons in plant expression can significantly improve the expression level of aminopeptidase gene in H.contortus.Further study indicated both Hc-ap-8 and Hc-ap-10 were expressed on the cell membrane of plant cells,but it is difficult to purify plant cell membrane proteins at present,thus,it is necessary to guide these two proteins to be expressed in the nucleus of leave cell.When codon-optimized Hc-ap-10(AP10^op)and non-optimized Hc-ap-8 were transiently expressed in tobacco leaves using the plant binary expression vector p H7LIC,they were also expressed in the cell membrane of tobacco leaf cells,but their expression levels were relatively higher than that in the p RI101 vector.Therefore,p H7d NLSAP8,p H7d NLSAP10^op and p H7d NLSHc23 with nuclear localization signal（NLS）SV40 were constructed using p H7LIC vector.When expressed in tobacco,codon-optimized Hc-ap-10showed higher expression level than unoptimized Hc-ap-8.However,it is surprising that the unoptimized Hc23 molecule can be expressed at higher level than optimized one,indicating the outcome of codon optimization can be affected by the size of targeted proteins.In the immunizing experiments of HC-AP-5 and HC-AP-10 proteins expressed in prokaryotic express system in goats,the combined use of these two antigens did not significantly reduce the infection burden of goat H.contortus since no significant difference were found in EPG and total worm burden between treatment and control group,indicating that the two prokaryotic Hc-ap-5 and Hc-ap-10 antigens are posibly not suitable candidate vaccine antigens for H.contortus.In conclusion,the present study constructed successfully the method to express protein in plant and the first time to express antigens from H.contortus in Nicotiana benthamiana,and meanwhile antigens,HC-AP-5 and HC-AP-10,expressed in E.coli could not significantly promote goats against infection of H.contortus.