Preparation Of Monoclonal Antibodies Against HCMV Important Epitopes

Human cytomegalovirus(HCMV),also known as human herpes virus type 5,is widely distributed in nature,and could cause a variety of diseases and most common congenital infection.HCMV presents a high infection rate worldwide,and only causes subclinical symptoms in healthy adults,who usually carry the virus in a latent rate.However,its infection or recurrence would lead to serious consequences in immunocompromised population.HCMV infection is found closely related to the pathogenesis of diabetes,cardiovascular diseases and malignant tumor.HCMV envelope glycoprotein B(gB)can induce neutralizing antibody.It can be detected in body fluids,such as urine and saliva,which suggests a promising prospect of gB in the diagnosis of HCMV infection.HCMV pp65 is a main structural protein of HCMV,which express in whole virus replication cycle,and it's highly conserved,which can also be used as an important biomarker for rapid diagnosis of HCMV infection.In presenting study,we planned to prepare monoclonal antibodies against important epitopes of HCMV gB and pp65.The coding sequence of gB neutralizing epitope(antigenic domains,AD)AD-1,AD-2,AD-3,was linked as AD 13 and biosynthesized in the company.The genomic DNA coding for pp65 dominant epitope region(297-510aa)was amplified by PCR.The target DNA fragments were ligated to pET-3 2a separately to construct recombinant prokaryotic expression vectors.The recombinant protein was expressed by IPTG induction and identified by Western blotting and ELISA.After washing of inclusion body,protein dialysis and renaturation,purification by His-taq chromatography and molecularsieve chromatography,the target proteins can reach a purity of 99%and be used for subsequent experiments.The 6-8 weeks BALB/C female mice were subcutaneously immuned with purified protein,and the antigen dose is 100 ?g per mouse per time.Serum of the immunized mice were collected to detect the titers of target antibody each time before immunization.After the forth immunization,OD450 of mice serum dilluted by millions of times could reach up to 1.0,and the mice were given a strengthening immuniazation via spleen and tain vein.3 days later,the spleen cells were collected for cell fusion with SP2/0 cells,and hybridomas were screened by 20%FBS-HAT-1640 cell medium.The positive hybridomas which secreting target antibody against AD 13 or pp65 were cloned by limiting dilution.Indirect ELISA and WB were used to analyze the immune reactivity and type of monoclonal antibodies secreted by positive cell strains.In summary,the recombinant AD 13 and truncated pp65 were successfully expressed in E.coli,and showed remarkably active immunereactivity and immubogenicity.Six hybridoma cell strains respectively secreting high reactive and specific monoclonal antibodies aganist AD 13 and pp65 were obtained,which layed a foundation for further developing HCMV vaccine and diagnositic reagent of independent intellectual property.