Generation And Identification Of Adsoption Inhibition Of Nanobodies Anti Rabbit Hepatitis E Virus Capsid Protein

Hepatitis E(HE)is a viral hepatitis caused by hepatitis E virus(HEV),which is an acute and self-limited zoonotic disease.Rabbits are the major natural host of HEV,maily responsbile for the transmission of genotye 3 HEV.Rabbit HEV can be transmitted to other animals and then get humans infected by food-borne or water-borne pathways.For this reason,blocking the transmission of rabbit HEV places an importance on the human public health.HEV contains three open reading frames(ORF),while ORF2 encodes capsid protein which has major epitopes related to the immune response involved in the main sites of vaccine developments.Besides that,virus like particle(VLP)based on the recombinant expression of ORF2 protein of HEV is widely used in the study of early entry process of HEV.The human genotype 1 HEV ORF2 truncated protein p239(368-606 aa)plays a major role in virus adsorption and invasion,meanwhile,p239 is able to induce body to produce high titer antibody for its high immunogenicity.Recently,swine HEV sp239(368-606 aa)protein adsorbing Hep G 2 cell model has been established successfully.Nanobody is a kind of heavy chain antibody(Hc Ab)which exists in the serum of camel family animals and naturally lacks the light chain as well as the first constant region CH1 of the heavy chain.The“nano”in the nanobody means its size is in nano scale.Nanobody has various advantages such as small molecular weight(15k Da),strong affinity,high stability,and easy production.With antigen immunized,the nanobody specific to immunogen can be obtained by phage screening technology which accelerates the application of nanobody in disease detection,diagnosis and treatment.This study expressed the truncated protein of rabbit HEV ORF2(368-606 aa)named r239 and established an adsorbing model of Hep G 2 cells,then the puritied r239 was used to immunize camel to prepare the nanobodies specific to r239 prorein.Finally,the effect of the nanobodies blocking r239 attaching Hep G 2 cells was identified in vitro.The key methods and results are listed below:1.Preperation and screening of r239 specific nanobodyTruncated protein r239 was expressed as inclusion body in E.coli system,purified by molecular sieve with the production of 60 mg/L.The purified r239 was immunized to bactrian camel for 6 times,2 mg each time.After the third immunization,the serum of camel was detected by indirect ELISA to evaluate the level of target antibody.After the fifth immunization,the specific anti r239 antibody titer reached 1:10~6.One week after the sixth immunization,collect the peripheral blood of bactrian camel and isolate the lymphocytes.The total RNA of the cells was extracted,then VHH gene was amplified by nested PCR using the c DNA as the template,to construct the recombinant phage library.Finally,the library with capacity of 3.5×10~8 pfu/m L was obtained from which the 11 anti r239 nanobodies were screened after 3 rounds panning by phage display technology.3.Prokaryotic expression,purification and functional verification of anti r239 nanobody Four high-affinity ones of the selected nanobodies were constructed into p ET21b prokaryotic vector for recombinant expression,then were purified by a nickel column with the fused his-tag.The results of SDS-PAGE,Western blot and indirect ELISA showed that the nanobodies were pure and specific.4.Determination of the optimal concentrations of the r239 used in adsorbing Hep G2 cells experimentsIn this part,the suitable concentrations of r239 in the adsorbing tests for different detectingmethods(Western blot,IFA and FCM)were determined by setting different gradients of r239concentrations.After identification,r239 can simulate the infection process of virus including adsorption as well as entering Hep G2 cells,and the optimal concentrations of r239 vary from the detecting methods.For IFA and FCM,0.4μmmol/m L r239 was quite enough to work well which was much lower than 2μmmol/m L used in Western blot to show great attachment.5.Inhibition of r239 adsorbing Hep G2 cells by nanobodiesFour purified anti r239 nanoantibodies(nb12,nb47,nb60 and Nb80)were mixed with r239of which the optimal amounts had been determined as described above,and the final concentration of nanobodies was incerased to 25μmmol/m L,in addition,rabbit HEV infected serum(1:100)served as a positive control and Nb-PEDV(specific to PEDV N protein)was used as a negative control.During Western blot and IFA,the mixture was incubated with Hep G2 cells at 4℃for an hour,while in the FCM the reacting time was prolonged to overnight.The results indicated that Nb47 and Nb60 could block r239 adsorbing Hep G 2 cells,which suggested the epitopes they recognized were related to attachment,showing the anti virus ability that they may have.In summary,this study established a model of a truncated HEV ORF2 protein r239adsorbing Hep G2 cells and expressed four anti r239 protein nanobodies,then verified three of them(Nb47 and Nb60)could partially block r239 adsorbing Hep G 2 cells.The generation of r239 nanobodies provides the principle of low-immunogenicity vaccine design as well as the control and treatments for HEV associated diseases.