Design And Preparation Of Yeast-based Herpes Virus Vaccine

Alzheimer’s disease(AD)is a neurodegenerative disease.It’s occurrence and development process are related to genetics,apparent modification,environment and other factors.There are many doctrines about Alzheimer’s disease,mainly including "choline dysfunction hypothesis","amyloid cascade hypothesis","tau hypothesis","viral infection hypothesis" and so on.The focus of research and development on AD drugs has been to eliminate the deposition of Aβ in order to improve patients’ cognitive dysfunction,but the successive failures of drug development have proved that this approach is not feasible.The "viral infection hypothesis" proposed in the past two years means that AD is caused by Herpes simplex virus 1(HSV-1)infection,and this hypothesis makes HSV re-enter people’s vision.Herpes virus can cause the deposition of Aβ and the hyperphosphorylation of tau,both of which are the main neuropathological features of AD.All along,it is generally believed that the senile plaques formed by Aβ deposition are the culprit of AD,so Aβ was once the target,but Aβ is also an antibacterial peptide,with antibacterial,antifungal and antiviral effects.Studies have shown that Aβ can wrap the herpes virus to protect neurons from damage.At present,the treatment of HSV-1 is to use some antiviral drugs,such as acyclovir,etc.But these antiviral drugs are only to relieve the pain of patients and reduce the frequency of occurrence,and can’t achieve the purpose of cure.Therefore,for HSV-1 infection,the most effective method is prevention,that is,vaccination.Preliminary laboratory research results show that protein disulfide isomerase(PDI)can increase the expression of foreign proteins in Pichia pastoris,so Pichia pastoris X-33-PDI that overexpressed PDI was used as a foreign protein expression strain to increase the expression of foreign protein.From the perspective of vaccines,considering that the current vaccines are mainly injection vaccines,and this vaccine requires protein purification during the research and development process,high cost,and must guarantee the entire cold chain during transportation,and it requires professional medical personnel to use it.So we used yeast surface display technology to construct yeast oral vaccines.The yeast display system contains three parts: M cellspecific ligand,which as a target;HSV-1 envelope glycoprotein D,pathogen protein,as antigen;α-lectin,anchored protein.After the yeast surface display strain was constructed,the location of the target protein was observed using a fluorescent microscope.Compared with the control group,green fluorescence was observed on the surface of experimental group strain,while the control group had no green fluorescence,proving that the target protein has been successfully expressed and displayed on the surface of yeast;At the same time,the expression of target protein was verified by western blot experiments.It was found that the target protein was detected in the wall of the experimental group,and the extracellular product did not contain the target protein,which again confirmed the target protein was expressed and localized on the yeast wall without leakage.And the target protein was not detected in the wall and extracellular products of the control group.After the above strains were induced and expressed,they were fed orally to BALB / c mice for the 1,3,5,7,9,11 weeks,and 5 days per week.Then regularly collect mouse blood samples and stool samples.Blood samples were collected by centrifugation to obtain serum.The stool samples were processed into homogenate and centrifuged to obtain supernatant.The specific antibody concentration was verified by ELISA.The results showed that the specificity antibody IgG of the blood samples,the specific antibody IgA of the stool samples gradually increased with time.Compared with the control group,the experimental group have significant differences,which proves that they can effectively cause humoral immune responses and mucosal immune responses.This for the HSV-1 vaccine development laid experimental foundation,is expected to be used for the prevention of herpes simplex virus infection.At the same time,it may open a new door for AD intervention.