Preparation Of Polyclonal Antibody Against HCMV UL147 And Evaluation Of The Efficiency Of Engineered RNase P Ribozymes Inhibit The Infection Of MCMV In Animals

Human cytomegalovirus(HCMV),the β subfamily of Herpesviridae of Herpesvirales,is a kind of virus that spreads widely in human beings.Most infections by HCMV are asymptomatic,but they may cause very serious consequences for immunocompromised patients,such as organ transplantation,AIDS patients,newborns,etc.There are many open reading frames in Human cytomegalovirus.They are not necessary for virus replication,but play an important role in immune escape.The UL146 and UL147 encoded proteins in the UL/b’ region of these open reading frames belong to the chemokine family,there are closely related to immune escape.The HCMV UL147 protein vCXCL2 belongs to the chemokine family CXC.In this study,we constructed a clonal expression vector by prokaryotic expression vector pET32 a,induced the expression of vCXCL2 to immunize New Zealand white rabbits to prepare polyclonal antibodies.The results were as follows:(1)The optimum conditions for inducing expression in BL21 strain: the optimum inducing temperature was 20 C,the inducing time was 6h,and the concentration of IPTG was 1 mM/ml.(2)Induced expression of recombinant fusion protein was eluted by nickel affinity chromatography with imidazole concentration of 100mM/L.The purity of SDS-PAGE was 98% by gray scale scanning;(3)Rabbit serum obtained by four immunizations was detected by ELISA,and its efficacy reached 100,000.Traditional drugs play an active role in the treatment of HCMV infection,however,the disadvantages are high toxicity and drug resistance.Numerous studies have shown that the engineered ribozyme P(M1GS)can effectively inhibit viral replication by blocking the expression of key viral genes.Engineering ribozymes R388-AS and M1-AS targeting MCMV AS gene were designed based on high catalytic activity mutant R388 and wild type M1.The protease encoded by the MCMV AS gene is important for the maturation of the viral capsid protein.Compared with wild type M1-AS,the catalytic activity of R338-AS was at least 200 times higher in vitro.In MCMV-infected cells,the expression of R338-AS inhibited viral AS was 98-99%,and the amount of progeny virus decreased by 15,000-fold.In the SCID mouse model experiment,the R338-AS activity was higher than that of wild type M1-AS,which can restrain AS expression to affect virus replication and improve survival rate of mice.