Cloning And Expression Of Monoclonal Antibody Gene Of Herpes Zoster Virus Glycoprotein E And Establishment Of Its Detection Kit

Varicella-zoster virus(VZV)infection can cause two diseases with different clinical manifestations: chickenpox(chickenpox)and herpes zoster(HZ).Chickenpox is a self-limiting disease,and the infected people are mainly infants and young children under the age of six,but the virus will be latent in the nerve cells of the infected person.It replicates in cells,leading to herpes zoster(HZ).The clinical manifestations are mainly characterized by fever,rash,and herpes.Complications include chickenpox pneumonia,encephalitis,and neurological sequelae,which can lead to death in severe cases.According to statistics,there are about 140 million new cases worldwide every year.However,domestic treatments and vaccines for varicella and herpes zoster caused by VZV infection are still under development.The VZV genome is about 125 kb in length and is a double-stranded DNA virus that can be translated to produce 67 proteins,including the main six glycoproteins(g E,g B,g H,g I,g C and g L).Among them,glycoprotein E(g E)is highly immunogenic and plays a major role in the virus infection of cells and the spread of viruses between cells.Studies have shown that immunizing animals with g E protein as an antigen can effectively stimulate the body’s immune response,thereby inducing the formation of antibodies.At this stage,recombinant subunit vaccines are prepared by using g E protein as an immunogen and adjuvant.Therefore,the research of detection kits based on g E protein is of great significance for the clinical detection and diagnosis of HZ.First,in this study,recombinant g E protein was used as an immunogen,mixed with adjuvant to immunize white rabbits,and the rabbit antiserum was purified by affinity chromatography.By polyacrylamide gel electrophoresis(SDS-PAGE),western blotting(WB)and indirect immunofluorescence assay(IFA),the results showed that the purified polyclonal antibody could specifically bind to g E protein.The polyclonal antibody coupled to HRP was used as the detection antibody,and the unlabeled polyclonal antibody was used as the capture antibody to construct a polyclonal-to-polyclonal detection kit.In this study,the accuracy,precision and specificity of the constructed detection kit were verified,indicating that the kit meets the standards and can be used for the detection of g E protein.Second,we used recombinant g E protein as the immunogen,mixed with adjuvant to emulsify,immunized mice,and obtained ten hybridoma cell lines stably expressing anti-g E antibody through cell fusion technology and subcloning screening technology.In this study,a large number of monoclonal antibodies were obtained by extracting and purifying mouse ascites by in vivo culture method.According to the methods of ELISA,WB and IFA,it was determined that the monoclonal antibody can bind to both recombinant g E protein and the native g E protein expressed by the virus.We expressed different fragments of g E through the prokaryotic expression system.The indirect ELISA results showed that different mAbs recognized different g E fragments,and a few mAbs recognized conformational epitopes.Afterwards,we performed an additive experiment on the ten successfully screened monoclonal antibodies,and finally determined that the purified antibody No.8 was selected as the coating antibody,and the No.6 antibody was selected as the detection antibody and labeled with horseradish peroxidase to establish a highly sensitive monoclonal antibody.Anti-monoclonal antibody ELISA detection kit.The mAb-to-mAb ELISA method established in this study can effectively detect VZV-g E protein and VZV virus,and can be applied to the rapid detection of virus and its g E glycoprotein later.Finally,we extracted total RNA from hybridoma cells,cloned the heavy chain and light chain of monoclonal antibody gene by RT-PCR technology,enzymatically linked with 18-T vector and sequenced.The comparison of the sequencing results confirmed that the monoclonal antibody gene obtained in this study was a specific monoclonal antibody.This study provides theoretical support for the future development of genetically engineered clinical therapeutic anti-g E antibodies.In summary,the anti-g E polyclonal antibody and monoclonal antibody were obtained by immune animals,and the multi-to-multi-antibody detection kit and the monoclonal antibody detection kit were prepared by using the obtained antibody.Since g E is the most abundant membrane protein in VZV expression,the kit we have constructed can not only be used as an effective detection method after VZV infection,but also achieve accurate quantification of g E protein,laying the foundation for the development of recombinant subunit vaccines in the later stage.