Expression Of Pseudorabies Virus Ge Protein And Screening Of Specific Nanobodies

Porcine Pseudoraibies(PR),also known as Aujeszky's disease,is an acute infectious disease caused by the(Porcine pseudorabies virus,PRV)belongs to the herpes virus family.Pigs are natural storage hosts and infectious sources of PRV,which can cause high mortality,nervous system disorders,reproductive disorders and respiratory diseases in infected pigs.In2011,outbreaks of swine pseudorabies were reported in various regions of China,which caused a huge loss in the pig industry.The prevention and control of swine pseudorabies has become a major issue in the current swine industry.Diagnosis plays a crucial role in the prevention and control of the disease.Therefore,establishing of new diagnostic method with high specificity and sensitivity is very important for the prevention and control of the disease.The gE envelope glycoprotein is highly conserved among PRVs,which is an ideal protein for diagnostic techniques.The nanobody has the following advantages:small molecular weight,high specificity,strong affinity,stable structure,high solubility,easy access to receptors,recognition of specific antigenic epitopes,expression in multiple systems,and low production cost.At present,nanobodies were widely used in clinical detection and diagnosis.Nanobody is a new antibody derivative,which have great prospects in Clinical application.In this study,camel was immunized with the purified gE recombinant protein.Then,a phage display library was constructed.Two specific antibodies against gE recombinant protein were Separated from the enriched library by phage display technology.These results provided the necessary experimental basis for establishing new diagnostic method of PR and the screening of nanobody with neutralizing activity.The main experiment content and results are as follows:1.Expression and Purification of gE Recombinant Protein:The recombinant plasmid PET-28a-gE was transformed into the competent cell Trans5?.The soluble gE recombinant protein was expressed after induction by IPTG.The gE recombinant protein was purified by Nie-column chromatography.SDS-PAGE analysis showed that the size of gE recombinant protein was 28 KaD.2.Immunization of Bactrian camel and construction of phage display libraryBactrian camels were immunized with the complex of 2mg purified gE recombinant protein and Freund's adjuvant.After the completion of the immunization,the specific antibody titer against the gE recombinant protein in serum was detected to be 1:256000.PBMCs were isolated from Peripheral blood.Total RNA was extracted from PBMCs.CDNA was obtained from total RNA by PCR.The amplified VHH gene was ligated to the pCANTAB 5E vector.The recombinant vector was transformed into TG1 competent cells by electroporation.The capacity of VHH phage display library was identified as 5×10~6.The positive rate and diversity of the library was identified by PCR and gene sequencing.The results showed that the positive rate was 99%,and the gene sequence displayed good diversity.3.Screening of specific nanobodies:The purified gE recombinant protein was used as a coating antigen and specific nanobodies were panned out.After three rounds of panning,the enrichment effect of specific phage was detected by ELISA.The results showed that the enrichment effect was obvious.Two specific antibodies against gE recombinant protein were finally screened out.In summary,the prokaryotic expression system was used to successfully achieve soluble expression of the recombinant gE protein.Using phage display technology to screen out specific nanobody against gE recombinant protein.It provides the experimental base for establishing new diagnostic method of PR and screening of nanobodies with neutralizing activity.