Construction And Validation Of Gluc-GFP Dual-labeled System And H5N1 Pseudovirus

Optical molecular imaging has important significance for the virus research. In the field of influenza virus, labeling of the viruses has developed quickly and greatly enhanced the study of pathogenesis of influenza virus and its infection process regulations. For some contagious, high pathogenic influenza viruses, the construction of their Pseudoviruses eliminates the security risks of operating live viruses. The excellent advantage of security and stabilityits of safety and stability of Pseudovirus make it an effective tool for research on viral pathogenisis and vaccine development and validation.The most commonly used molecular imaging methods in biological research are fluorescent molecular imaging(Fluorescence) and bioluminescence imaging(Bioluminescence). They both have their advantages and disadvantages. Bioluminescence has no autofluorescence, with high specificity and sensitivity characteristic, can be used for accurately quantification. Its shortcoming is the weak signal, the longer detection time and higher cost. Fluorescence molecular imaging pocess strong signal, quickly imaging process, low-cost and stability. However, it is difficult to be accurately quantified due to its limited depth and prohibit its in vivo applications. In this study, the Green fluorescent protein(GFP) and Gaussia luciferase(Gluc) were selected for double molecular imaging. This combines their advantages and overcome their deficiencies.H5N1 avian influenza virus is a highly pathogenic influenza virus. It spreads fast and has high mortality. For the highly pathogenic virus, high-level biosafety laboratories are needed for live virus operation. This requirement increases the cost and difficulty of the experimenst. The construction of the H5N1 Pseudovirus can overcome its biosafety risks. H5N1 Pseudovirus provides a safe and effective tool for the researchof H5N1 virus pathogenicity and the development of antiviral drugs.Split-GFP gene was synthesized according to the documented reference. GFP was separated into G1-10 and G11 and inserted into pc DNA3.1 expression plasmid, respectively. Their interaction was tested by fluorescence observation. Then fusion PCR was performed to link Gluc gene and G11 gene and inserted the fused gene into pc DNA3.1. After co-transfection of the fused plasmid and G1-10 plasmid to the 293 T cells, the expression of luciferase in the supernatant was measured and fluorescence was observed. To simplify the construction process, MDCK cell line consistently express G1-10 was constructed by Lentivector Expression Systems. Then G11 plasmid was transfected to G1-10 MDCK cell line and puromycin and RFP were employed for selection and validation of the dual-labeled system. The fluorescence observation confirmed the feasibility of split-GFP strategy. After co-transfection of G1-10 and Gluc-G11 plasmids, expression of luciferase in the supernatant could be measured and fluorescence could be observed. The constructed G1-10 MDCK cell line could stably express G1-10. This study successfully constructed and validated the Gluc-GFP dual-labeled system. The physiological activity of Gluc was not affected by linking G11, stable G1-10 MDCK cell line could simplify the construction of the dual-labeled system. This dual-labeled system provided a powerful tool for further investigating the pathogesis of viruses.Furthermore, the pc DNA3.1-HA plasmid containing H5N1 HA gene was constructed and co-transfected with p NL4-3.Luc.RE into the 293 T cells. The expression of luciferase in the supernatant was measured after 72 h. Viral supernatants were then purified by PEG-it and the purified H5N1 Pseudovirus was identified by ELISA experiments using H5N1-HA antibodies. The results showed that the cell supernatants containing the expressed luciferase and the expression of HA can be detected by ELISA experiment. This confirmed the success construction of H5N1-HA pseudotyped virus.