Prokaryotic Expression Of Human Cytomegalovirus GH Protein And Preparation Of Its Monoclonal Antibody

Human cytomegalovirus(HCMV)is a kind of herpesvirus which infects people all over the world,and it is also the most common pathogen that causes fetal congenital malformations and death of organ transplant recipients.Based on the social and economic burdens and the lack of effective treatments,HCMV has been designated as one of the most priority vaccine targets,and the development of its neutralizing antibodies can lay the foundation for the preparation of preventive or therapeutic HCMV vaccines.g H(glycoprotein H)is encoded by UL75 of HCMV,with a total length of 2 232 bp and 742 amino acids.g H,g B and g L constitute the core fusion mechanism of HCMV,which plays a key role in the process of HCMV infection of host cells,and is the main target site for inducing immune response.Some studies have shown that g H was the main neutralizing antigen of human cytomegalovirus,and specific anti-g H antibody had the potential to neutralize HCMV and block the transmission of virus between cells.The purpose of this study was to obtain specific anti-g H monoclonal antibodies.The BAC containing the whole genome of Toledo,a clinical strain of human cytomegalovirus,was used as a template to amplify the g H gene fragment without signal peptide and transmembrane region,Then the g H gene fragment with a total length of 2 064 bp and encoding for 688 amino acids,was inserted into the expression vector p ET32a’,to construct the recombinant expression plasmid of p ET32a’-g H.The constructed plasmid with correct sequence was transformed into the expression strain Trans B,and recombinant g H protein at a mass of 78 k D was successfully expressed by the induction of IPTG,which was mainly expressed in the form of inclusion body.The purified g H protein was obtained by inclusion body washing,urea denaturation and electroelution.Eight-week-old male BALB/c mice were immunized with purified recombinant g H at a dose of 150 μg every time.Indirect ELISA was used to detect the serum titer of mice one day before each immunization.After five times of immunization,the serum titer of mice reached at 1: 100 000,indicating that the expressed g H protein had good immunogenicity.Under aseptic condition,the spleen cells of immunized mice were collected and then fused with SP2/0 myeloma cells.After four rounds of subclonal screening,five cell lines stably secreting anti-g H monoclonal antibodies(m Abs)were obtained,which were respectively named as 8A9,8B4,8C4,8D9 and 8D12.All of the five monoclonal antibodies had good reactivity to g H protein.Among the five m Abs,8A9,8C4,8D9 had higher affinity,while 8D9 and 8D12 had stronger ability to capture virus.In present study,we obtained several specific anti-g H monoclonal antibodies with independent intellectual property rights.The antibodies presented a good ability to capture virus,which will lay the foundation for the development of neutralizing antibody to prevent or treat HCMV infection in the future.