Preparation,purification And Identification Of Polyclonal Antibody Against HCMV PUL147A

Objectives:HCMV assigned to theβherpesvirus family can easily infect the majority of the world’s population.In immunocompromised suffers,some complications are associated with HCMV reactiviation developed from latent infection in healthy carriers.Seriously,it can lead to disturbances in the body function and even death.It is still unclear about HCMV pathogenesis,but extensive research involved in the UL/b’genic polymorphism revealed that the UL/b’is the genetic basis of HCMV pathogenicity.The UL/b’contains 19 genes ranging from UL133 to UL151.According to the significance of the UL/b’to HCMV pathogenesis,it can be inferred that UL147A included in the UL/b’may play an important role in HCMV replication,latency and pathogenicity.Furhermore,it may also affect multiple immunomodulatory mechanism of host.However,there are few reports about the functions of UL147A.Therefore,in this study pUL147A polyclone antibody which can contribute to the discovery of the stucture and function of UL147A was prepared for laying a foundation for intensive research about HCMV pathogenesis.Methods:This study consists of two parts:(1)Expression and purification of UL147A recombinant protein:UL147A was amplified with a template HCMV Towne EL-350 and cloned into a vector pET32a(+).Once the recombinant plasmid UL147A-pET32a(+)established,transforming it into the strain DH5αfor clone and then into the strain BL21(DE3)for expression after a series of experimental identification.Optimizing the expression of UL147A recombinant protein induced by IPTG,time and temperature and then collecting a great quantity of UL147A recombinant protein.Later,the highly purified UL147A recombinant protein was obtained through Ni-NTA affinity chromatography in optimal conditions.(2)preparation of pUL147A polyclone antibody:To prepare the specific polyconal antibody,New Zealand rabbits were immunized by the highly purified UL147A recombinant protein mixed with an adjuvant.The titer of the polyconal antibody was evaluated by ELISA untill it reached to 10~5,subsequently,the immune serum was extracted and then purified by ammonium sulfate precipitation and affinity chromatography.Finally,the sensitivity and the specificity of the pUL147A polyclone antibody were determined by ELISA and Western blot assays.Results:(1)Expression and purification of UL147A recombinant protein:The recombinant plasmid UL147A-pET32a(+)was successfully established and UL147A recombinant protein was also can be expressed in the strain BL21(DE3).The target protein expressed as inclusion body was optimized as follows:the concentration of IPTG was 1 mM,the time was 10 h and the temperature was 25℃.UL147A recombinant protein was highly purified by elution buffers which contain imidazole in different concentrations,and the optimal concentration of imidazole in elution buffer was 150 mM.(2)preparation of pUL147A polyclone antibody:The titer of the immune serum purified was over 10~5,which was evaluated by ELISA.And Western blot assay suggested the high specificity of pUL147A polyclone antibody.Conclusion:(1)UL147A recombinant protein was successfully expressed in prokaryotic expression system and highly purified.(2)The highly purified and specific pUL147A polyclone antibody was prepared.