Title Page
Contents
Abstract 8
Introduction 10
Materials and methods 13
2.1. Cell 13
2.2. Virus and plaque assay 14
2.3. Exploration of the eGFP fluorescence wavelength of rVHSV 15
2.4. MOI and culture conditions for optimal screening 17
2.5. Optimization of floating condition for dispensing process 18
2.6-1. Substances for screening 19
2.6-2. Screening procedures 20
2.7. Second-round screening of selected substances 22
2.8. CC50 and MNCC for cytotoxicity of selected candidates[이미지참조] 23
2.9. Determination of IC50 of final candidates against VHSV[이미지참조] 25
2.10. Preparation of extract from different part of Atrina pectinata 26
2.11. Antiviral activity test of extracts from different parts of A. pectinata against VHSV 28
Results 29
3.1. Optimization of rVHSV eGFP fluorescence wavelength 29
3.2. Optimization of MOI, culture condition for screening 31
3.3. Determination of optimal speed for mixing 33
3.4. Screening of antiviral substances 34
3.5. Second-round screening with effective concentration 36
3.6. Cytotoxicity of selected candidates 40
3.7. Antiviral activity of final candidates 43
3.8. Anti-viral activity of extracts from different parts of A. pectinata. 46
Discussion 48
국문 초록 52
Reference 54
Table 1. Wavelengthsettings for eGFP measurement from rVHSV inoculated EPC cells 16
Table 2. Identification of two chemical substances as final antiviral candidates. 39
Table 3. Identification of two final two extracts as final antiviral candidates. 39
Figure 1. General procedures of antiviral mass screening by using recombinant VHSV as a surrogate virus. 21
Figure 2. Photos of A. pectinata and parts of A. pectinata used in ultrasonic extraction experiments. 27
Figure 3. Optimal fluorescence wavelengthsearch for rVHSV eGFP measurement. 30
Figure 4. Optimization of culture temperature, FBS concentration and MOI for the mass screening of antiviral agents with recombinant VHSV. 32
Figure 5. The fluorescence range of 27 substances selected from the primary screening. 35
Figure 6. Morphological changes of EPC cells infected with rVHSV and treated with different concentrations of chemical candidates from primary screening. 37
Figure 7. Morphological changes of EPC cells infected with rVHSV and treated with different concentrations of extract candidates from primary screening. 38
Figure 8. Cell viability cells treated with selected chemical compounds A and B for the determination of CC50.[이미지참조] 41
Figure 9. Cell viability cells treated with selected extracts EA and EB for the determination of CC50.[이미지참조] 42
Figure 10. Inhibition of rVHSV infectivity by selected chemicals. 44
Figure 11. Inhibition of rVHSV infectivity by selected extracts. 45
Figure 12. Antiviral activity against rVHSV of extracts from different parts of A. pectinata. 47