Amored RNA And Its Application In Prevention And Control Of Transfusion-transmitted Disease

Blood safety has been improved along with the application of the new technique and the implement of the policy of the non-remunerate volunteer blood donation. But the residual risks of transfusion-transmitted diseases are still inevitable. Although the development of nucleic acid test (NAT) blood screening and the application of the pathogen inactivation will further reduce the residual risk of blood transfusion, both of these techniques need a stable, safe and effective control in practice.Armored RNA technology put the sequences of MS2 and exogenous sequences into the expression plasmid together, and can express the armored RNA containing the exogenous sequences in vitro. Since the coat can protect inside RNA against RNase, The armored RNA can be used as a stable, safe and effective control in NAT blood screening and pathogen inactivation.In this study, armored RNA technology platform was established. The sequences of the assembly protein, coating protein and PAC sequence were cloned into a expression plasmid, and the armored RNA was transcripted and expressed in vitro. Then the exogenous sequences were cloned into the armored RNA platform and the armored RNA containing the exogenous sequences was expressed. The results showed that the amored RNA containing both 200bp and 1400bp completed inserted exogenous fragments were expressed. Meanwhile, the expressed amored RNA remained the stability as same as MS2 bacteriophage, since the outer envelope can effectively protect the internal MS2 and exogenous fragments to be degraded by RNA enzymes. So compared to the live virus, the amored RNA is a none-proliferative and more secure control without infectious ability. And compared to the RNA fragments, it has better stability and cannot be degraded by RNA enzymes easily.The feasibility of the amored RNA as a control in NAT was discussed firstly. The amored RNA containing the 440bp HCV fragment and another amored RNA containing HCV, HIV and HBV complex sequences were both expressed and were tested with PIJI and KEHUA commercial kits, respectively. The results showed that the amored RNA can effectively simulate the existence of live virus, and it has better stability and security compared to live virus and RNA transcripted controls.Secondly, the feasibility of the amored RNA as an evaluation panel in pathogene inactivation validation testing was discussed. The amored RNA containing the 1400bp Sindbis virus fragement was expressed in the technologic platform, and were inactivated by methylene blue photochemical methods with the control of the live Sindbis virus at the same time. Then, the relationship between the infectious ability of the live virus and the RNA damage were analyzed. The results showed that the infectious ability of the live virus was gradually decreased with the photochemical inactivation treatment, while its RNA damage was gradually increased. By analyzing the results, the decrease tendency with the log value of the nucleic acids of live virus was linear correlation with that of the infectious ability of the virus; On the same time, the damage of armored RNA was also increased with the photochemical inactivation treatment. And the decrease tendency with the log value of the nucleic acids of amored RNA was linear correlation with that of the live virus. From the results, it can be concluded that the amored RNA can be used as an evaluation panel to assess the validation of the photochemical inactivation treatment with real-time PCR.The amored RNA not only can be used as the safe, stable and effective control in NAT, but also can be used as the evaluation panel to assess the validation of the photochemical inactivation treatment with real-time PCR. It has been believed that with further development and application, the amored RNA technique and it product can be widly used in the control and prevention of transfusion-transmitted disease.