신종인플루엔자 대유행 대비 백신 라이브러리 제작 및 백신 후보주 효능 평가

초록

Purpose : One of the most important issues for preparedness of global crisis of pandemic influenza is to harbor an appropriate vaccine candidates which can be use for timely vaccination strategy. To prepare vaccine candidate, reverse genetic(RG) system is used world-widely. For the RG system, Hemagglutinine(HA) antigen and Neuraminidase(NA) antigen are major determinants for antigenicity of the vaccine. In this study, we are trying to develop and characterize genomic libraries of avian influenza virus, which can be rapidly used for RG system when pandemic situation is being occurred. Methods : Highly pathogenic avian influenza virus(HPAI) A/H5N1-ES03 and A/H5N1-IS06 which were isolated 2003 and 2006 outbreaks in Korea, respectively, were use as target viruses for preparation of libraries. A/PR/8/34 virus was also used for hiring donor genes including PB1, PB2, PA, NS, NP and M in reverse genetic system. To remove highly pathogenic RKKR(amino acids) site from HPAI, PCR cloning technique with mutant primers was used. In addition, the 5' random amplification of cDNA ends(5'-RACE) was set up to analyze fine structure of 5'- and 3' proximal ends of each gene. Baculovirus expression system was recruited for expression of HA antigen from HPAI viruses and its antigenicity was analyzed. Results : 16 genomic libraries from HPAI viruses and 6 libraries from donor genes were constructed into TA cloning vector and their sequence identities were determined. After removing HP site, sequencing results revealed that RKKR multi-basic amino acids were successfully removed. To approach methodologically, 5' RACE was set up and no significant change of 5'- and 3'- proximal ends was observed when genes isolated from seasonal influenza viruses were analyzed. This method will be applied to analysis fine structure of genes from H5N1 viruses in successive research year. In addition, 64-70kDa recombinant protein was obtained from baculovirus expression system and this protein will also be used for antigenic characterization of HPAI viruses. Conclusions : Through the 1st period of the study year, we could obtain recipients and donor influenza genomic libraries and their sequence information. These results will be applied to prepare recombinant viruses using RG system and antigenic and genetic characterization of HPAI viruses in following period of study year.

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