JEV C RNA 11kb 3. Japanese encephalitis virus JEV JEV C 7. C E prm C 1 RNA. Gold pgbkt7 pgadt7- T pgbkt7-53 pgbkt7- JEV C PCR JEV C cdna

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1 CHINA TROPICAL MEDICINE Vol.12 No.4 April * JEV C PCR JEV C cdna pgbkt7 EcoRI/BamHI PEG/LiAc pgbkt7- JC pgbkt7 Gold Western- blotting BD- JC pgbkt7- JC Genebank JEV SA JEV C pgbkt7- JC pgbkt7 Gold SD/- Trp SD/- Trp/X SD/- Trp 30 OD600 >0.8 SD/- Trp/X/A Western- blotting 35KDa BD- JC pgbkt7- JC BD- pgbkt7- JC R A Construction and verification of a bait vector for the gene encoding core protein derived from Japanese encephalitis virus. DING De- ping FENG Guo- he. Department of Infectious Diseases, Affiliated Shengjing Hospital of China Medical University, Shenyang , China Abstract Objective To construct a bait vector with the gene encoding core protein derived from Japanese encephalitis virus,toxicity and self- activation of the bait protein were tested. Methods Full- length gene of JEV C protein was amplified by PCR method. The identified PCR product was inserted between EcoRI and BamHI sites of pgbkt7 vector The bait vector pgbkt7- JC was confirmed by restriction endonuclease enzyme analys and sequencing; The pgbkt7- JC and pgbkt7 were transformed into their respective yeast strain Glod cells by PEG/LiAc method.toxicity and self- activation of the bait protein was tested by cultured in SD with different amino acids defects and the phenotype assay. The expression of the bait protein BD- JC was detected by western- blotting. Results The gene fragment of JEV C protein was amplified and cloned into pgbkt7 successfully and its sequence was the same as that of the published JEV SA strains in the Genebank. pgbkt7- JC and pgbkt7 were transformed into their respective yeast strain Glod cells as well, both groups those cells only grew in SD/- Trp SD/- Trp/X but not in SD/- Trp/X/A indifference and the colours were not turn blue,and the OD600 were more than 0.8 by cultured overnight at 30 in SD/- Trp fluid medium in both groups cells. The size of BD- JC protein expressed in Glod cells were transformed with pgbkt7- JC was 35KDa. Conclusion The bait expression vector of pgbkt7- JC was constructed successfully,neither toxicity nor self- activation of the BD- JC protein expressed in Glod cells were found,which layed the foundation for screening and proving target proteins interacting with pgbkt7- JC using the yeast two- hybrid technique. Key words Japanese encephalitis virus Two- hybrid system techniques Vector construction Bait protein Japanese encephalitis virus JEV JEV C RNA 11kb 3 JEV C 7 3 C E prmc 1 RNA 1.1 pmd- JC [1] Gal4 3 JEV C Gal4 3 Gold pgbkt7 pgadt7- T pgbkt7-53 pgbkt7- No.L ~ * E- mail fenggh@sj- hospital.org

2 428 CHINA TROPICAL MEDICINE Vol.12 No.4 April Lam X- α- gal SD/- Trp 30 3~5d Aureobasidin A AbA DNA- BD Clontech JM SD/- Trp PCR pgbkt7- JC pgbkt7 EcoRI/BamHI DNA DNA T4 Marker TaKaRapGBKT7- JC pgbkt7 PVDF - 5ml SD/- Trp rpm IgG Santa PCR PCR Genebank AF SA SD/- Trp JEV C PCR pgbkt7- JC EcoRI 100ul SD/- Trp BamHI PCR 5 - CCG SD/- Trp/X- α- gal SD/- Trp/X SD/- Trp/ X- α- gal GAA TTC ATGACTAAAAAACCAGGAGGGC- 3 / AbA SD/- Trp/X/A CGC GG ATC C A GGCTCCTGCACAA 3~5d GCTATGA- 3 96~ bp SD/- Trp/X PCR SD/- Trp/X/A pmd- JC200ng 5 PrimeSTAR R Buffer Mg2+plus 10μl dntp Mixture 2.5 mm 4μl 10μM PrimeSTAR R Polymerase 2.5 U/μl HS DNA 0.5μl 50μl PCR 98 10s 55 10s 72 1min minPCR 1% SD/- Trp pgbkt7- JC pgbkt7- JC rpm OD600 DNA PCR 0.4~ g 4 2min EcoRI/BamHI 37 15h 2ml 20ml SD/- Trp 3h OD600 OD600>0.8 pgbkt7-53 pgadt7- T pgbkt7- Lam pgadt7- T SD/- Trp/X/A Western- blotting pgbkt7 3ml SD/- Trp 1:1 PCR 2 SDS- PAGE 100 pgbkt7 3h DNA 10min 20ul SDS- PAGE PCR 15V 20min 5% 37 2h 0.3pmol 0.03pmol DNA T4 BD- mab 4 IgG JM109 1h 30μg/ml LB pgbkt7- JC pgbkt7 37 BD- JC BD 1μg 2 EcoRI/BamHI 1% 2.1 JEV C PCR T7 PCR JEV C JEV C pgbkt7- JC cdna 381bp TE/LiAC 2.2 pgb KT7- JC Gold PEG/LiAC EcoRI/BamHI 381bp 7.3kb pgbkt7- JC pgbkt7 2 Gold T7 Genebank

3 CHINA TROPICAL MEDICINE Vol.12 No.4 April AF SA JEV C pgbkt7- JC MEL1 α pgbkt7- JC SD/- Trp/X/A JEV C 2. DL2000 DNA- Marker 1. the gene encoding JEV C protein 2. DL2000 DNA- Marker 1 JEV C PCR 1% Fig 1 The amplified PCR products of the gene encoding JEV C protein were detected by agarose gel electrophoresis. 381bp 7 300bp 381bp BD- JC AUR1- C AbA AbA AbA AbA pgbkt7-53 pgadt7- T Western- blotting pgbkt7 22KDa pgbkt7- JC 35KDa JEV C BD Gold BD- JC BD 22KDa JEV C 13KDa pgbkt7 pgbkt7- JC pgbkt7- JC 2. 1kb plus DNA ladder Marker 2 pgbkt7- JC EcoR I/BamH I 1% Fig 2 The plasimd of pgbkt7- JC was digested with restriction endonucleases enzyme EcoR I/BamH I and detected by agarose gel electrophoresis 2.3 pgbkt7- JC pgbkt7 pgbkt7- JC pgbkt7 Gold SD/- Trp 30 3~5d transformed with pgbkt7-jc Glod cells were transformed with pgbkt7 pgbkt7- JC pgbkt7 4 Western- blooting Gold Fig 4 Western- blooting analysis of proteins 2.4 SD/- Trp pgbkt7- JC pgbkt7 3 pgbkt7- JC pgbkt7 SD/- Trp OD BD- JC pGBKT7-JC BD-JC 2. pgbkt7 BD BD-JC protein expressed in the yeast strain Glod cells were 2. BD protein expressed in the yeast strain 3. Protein expressed in the yeast strain Glod cells were not transformed with any plasmid Gal4 3 Gal4 3 pgbkt7 2.5 pgbkt7- JC DNA Binding Domain BD SD/- Trp/X SD/- Trp DNA pgadt7 X- α- gal α- Activation Domain AD 35KDa 22KDa GAPDH

4 430 CHINA TROPICAL MEDICINE Vol.12 No.4 April DNA DNA Gal4 3 JEV C JEV C BD AD MEL1 AUR1- C JEV 1 Ding DP [3] Epidemiology and Infectious Disease JEV C Chinese [4] 2011, [5] JEV C 2 Williamson MP Biochemical Society transactions Ghosh D Gal4 3 - perspective J. PLoS neglected tropical diseases 2009 [6] Gal4 3 4 Chen SO Fang SH Shih DY JEV C response J. Virus genes : JEV C 5 Mori Y Okabayashi T Yamashita T Journal of Virology pgbkt7- JC Gold techniques and current limitations J. Plant Journal Feng GH. The interaction between Japanese encephalitis virus proteins and host proteins J. International Journal of Diagnosis and treatment of a pulmonary Cryptococcosi case. CHEN En- bin. Hainan Frontier Garrison of Armed Police Haikou Hainan P. R. China In. J. Sutcliffe MJ. Protein -protein interactions J. Basu A. Japanese encephalitis-a pathological and clinical 3 9 e437. et al. Recombinant core proteins of Japanese encephalitis virus as activators of the innate immune et al. Nuclear localization of J apanese encephalitis virus core protein enhances viral replication J. 6 Lalonde S Ehrhardt DW Loqué D et al. Molecular and cellular approaches for the detection of protein -protein interactions latest !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! R519.4 B Pulmonary Cryptococcosis d % ~

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