Study On Identifying Of Gene Expression Patterns Of EBV And Construction Of Lentiviral Vector Containing EBV-LMP2A Gene

It is significant that identify the status of virus-host interaction in patients with EBV infection early thereby providing information for effective therapy. Loop-mediatedisothermal amplification (LAMP) is a new method has been increasingly applied to rapidly diagnose infection by microbial pathogens and other clinical disease. On the principle of strand-displacing DNA synthesis by the Bst DNA polymerase, the LAMP technique using four pairs of primers to recognize six distinct regions of the target sequence. Under the same isothermal conditions, at a range of 60-65? without sophisticated apparatus, and one-step RT-LAMP can be completed within 1 h or less. LAMP is specific, sensitive, short time-consuming and simple operating, and the results can be interpreted with inspection by the naked eye by examining either turbidity or fluorescence.In recent years, EBV associated liver disease have been subsequently reported. The factor of chronic liver diseases is variety and the pathogenesis is complex. In China, hepatitis viruses are the major cause of chronic liver diseases, for example, Hepatitis B virus (HBV) and hepatitis C virus (HCV) infect millions of people and induce liver diseases. In addition, there is some correlation between EBV and liver damage or liver cirrhosis. We have a lot of research on hepatitis virus infection in patients with liver disease, but lack of the studies on EBV infection. However, make it clear that the preverlance of EBV in patients with liver disease can aid the early prevention and the prompt treatment of chronic liver diseases.LMP2A maintains viral latency by mimicing tonic signaling from the B-cell receptor (BCR), which is essential for latently infected B cells survival. But the study of LMP2A is unclear. Recently, researchers established EBV vaccines on LMP2A which is an ideal target antigen for EBV associated malignancy.Three parts of this study is closely related to each other. Our innovative results were presented as follows:1. Here we established a series of RT-LAMP assays for examining the expression of EBNA1, EBNA2, LMP1, LMP2A and BZLF1. EBV latent/lytic transcript were detected by RT-LAMP using the corresponding primers, the reaction at 63? for 45 min can have the effect amplification. The calcein can be added into the tube before the reaction, thus results can be visually assessed without opening the tube. RT-LAMP reaction conditions were optimized, including concentration of the betaine concentration, dNTPs concentration, magnesium ions, reaction time, reaction temperature, etc. And the specificity and sensitivity of RT-LAMP were tested. The results indicated that the RT-LAMP assays of EBV latent and lytic transcripts have high specificity. RT-LAMP was capable of detecting concentrations as low as 0.01% EBV-positive cells, which was equivalent to the sensitivity of RT-qPCR, but 100-fold higher than conventional RT-PCR. These assays may be employed in further investigations because they can aid the design of improved therapeutic regimens and can be used specifically in resource-poor settings.2. This study evaluated the prevalence of EBV in 187 specimens from patients with chronic liver disease. Four marked antibodies, which namely anti-VCA (viral capsid antigen) IgM and IgG, anti-EA (early antigen) IgG and anti-EBNA (EBV nuclear antigen) IgG, were investigated using ELISA. According to the test results of four different antibodies, the total samples enrolled are further grouped as primary infection, past infection or no infection. Combined with EBV DNA sample validation and analysis of clinical data, we found that EBV often presented latent infection and past infection in most cases. This result suggested that reactivation of EBV may be linked with liver damage. The expression pattern of EBV transcripts were identified using our established RT-LAMP, and we found that it presented latency ? pattern (expressed EBNA 1, LMP1 and/or LMP2A) or a pattern similar to latency ? (expressed EBNA 1, EBNA2, LMP1 and LMP2A or expressed EBNA 1, EBNA2 and LMP1) in patients with chronic liver disease. 3. We constructed pHAGE LMP2A lentiviral expression vector and pLKO.1-LMP2A shRNA gene-silencing vector, which provided research foundation and reference value for the further study of LMP2A gene function and further investigation on the therapy vaccine against EBV associated malignancies.