Microsoft Word - 12栾世家.doc

Transcription

1 生物工程学报 Chin J Biotech 2009, May 25; 25(5): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 医学与免疫生物技术 A NS1 栾世家 1,2, 潘蔚绮 1,3, 李婷 1,3, 杨化强 1, 张北武 1,3, 李锋 1,3, 陈凌 1, , , : 根据 A 型流感病毒密码子使用偏嗜性, 选取稀有密码子对 A/Puerto Rico/8/34(H1N1) 病毒 NS1 基因内部 110 个氨基酸区域进行密码子同义突变改造, 并全基因合成 NS 基因, 利用反向遗传操作技术拯救出含有密码子去优化 NS1 基因的重组病毒 (deons) 体外细胞噬斑形成实验和病毒生长曲线证明该病毒在 MDCK 细胞内的感染和复制能力比野生型病毒低约 1000 倍 ; BALB/c 小鼠体内致病力实验证明 deons 病毒不能引起小鼠发病和死亡, 该病毒在小鼠肺内的复制滴度比野生型病毒低 100~1000 倍 本研究探索了通过基因组密码子去优化改造途径降低 A 型流感病毒毒力的可行性, 首次证明流感病毒 NS1 基因密码子去优化同义突变可以降低病毒毒力, 为流感减毒活疫苗的研究提供了新的思路 : A 型流感病毒, NS1 基因, 密码子去优化, 致弱, 减毒活疫苗 1,2,3 Rescued influenza A virus with codon deoptimized NS1 gene is attenuated both in vitro and in vivo Shijia Luan 1,2, Weiqi Pan 1,3, Ting Li 1,3, Huaqiang Yang 1, Beiwu Zhang 1,3, Feng Li 1,3, and Ling Chen 1,2,3 1 Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences, Guangzhou , China 2 Department of Biomedicine and Biotechnology, University of Science and Technology of China, Hefei , China 3 Guangzhou Institute of Respiratory Disease, National Key Laboratory of Respiratory Disease, Guangzhou , China Abstract: To develop novel live attenuated influenza vaccine, we explored the feasibility to attenuate influenza virus by codon deoptimization of NS1. According to the codon usage bias in influenza A virus, we designed and synthesized a condon-deoptimized NS gene by substituting codons of 110 amino acids in the NS1 gene of A/Puerto Rico/8/34(H1N1) with unpreferred synonymous codons. The influenza A virus with the codon deoptimized NS1 gene (deons virus) was rescued by reverse genetics. Plaque forming assay and virus growth curve showed that the growth of deons virus was reduced about 1000 times in MDCK cells compared to that of the wild-type virus. Intranasal inoculation with deons virus did not cause death or evident disease in infected BALB/c mice. Furthermore, the virus titer in the lungs of mice infected with deons virus was significantly lower (i.e times) than that of wild-type virus. Our results indicated that influenza virus could be effectively attenuated by synonymous codon deoptimization of Received: January 23, 2009; Accepted: March 13, 2009 Supported by: Guangzhou Science and Technology Program (No. 2006E1-E0011). Corresponding author: Weiqi Pan. Tel: ; Fax: ; pan_weiqi@gibh.ac.cn Ling Chen. Tel: ; Fax: ; chen_ling@gibh.ac.cn 广州市科技计划项目 (No. 2006E1-E0011) 资助

2 : A NS1 721 NS1 gene. This strategy will be useful to develop new attenuated candidates for the production of live attenuated influenza vaccines. Keywords: influenza A virus, NS1 gene, codon-deoptimization, attenuation, live influenza vaccine A,,,,,,,,,,, [1 3],,, [4 8] NS1, I,, Garcia-Sastre [9] NS1 A [10], NS1, NS1,, NS1, 1 材料与方法 1.1 细胞 质粒和载体 MDCK 293T, 10% DMEM ppoli pcaggs Peter Palese A/Puerto Rico/8/34(H1N1)(PR8) (ppoli-pr8pb2 ppoli-pr8pb1 ppoli-pr8pa ppoli-pr8ha ppoli-pr8np ppoli-pr8na ppoli-pr8m) (pcaggs-pb2 pcaggs-pb1 pcaggs-pa pcaggs-np) 1.2 病毒和实验动物 12 A/Puerto Rico/8/34(H1N1) (R-PR8) SPF, 6 BALB/c 1.3 主要试剂 Sap I NEB, Trizol Lipofectamine 2000 Reagent Invitrogen, TPCK-Trypsin AEC Chromogen Kit Sigma, DNase I(RNase free) SYBR PrimeScrip RT-PCR Kit ( ), RNase A(DNase free) MBI, Viral RNA Extraction Kit Qiagen, A NP Virostat, IgG 1.4 密码子去优化 NS 基因的设计与合成 NS [11] NS1 NS2 [12], PR8 NS1 151~ , A [10], (Codon deoptimization), NS 5 3 ppoli Sap I, deoptimized NS, ( ), PUC ppoli-deons 转录质粒的构建及制备 Sap I puc57-deons,, 900 bp ; Sap I ppoli, T4 DNA 16 o C, Top10, ppoli-deons

3 722 ISSN CN /Q Chin J Biotech May 25, 2009 Vol.25 No 病毒拯救和鉴定 ppoli-deons 7 ppolis(pb2 PB1 PA HA NP NA M) 0.5 μg, 4 pcaggs(pb2 PB1 PA NP) 1 μg, lipofectamine T MDCK 37 o C 5% CO 2 8 h,, 1 ml Opti-MEM( 0.5 μg/ml TPCK-Trypsin), 48 h,, 4000 r/min 2 min, 0.2 ml 9 SPF, 37 o C 48 h,, WHO [13],, deons Trizol RNA, DNase I, Uni-12(5 -AGCAAAAGCAGG-3 ) cdna, NS (NS-1:5 -AGCAA AAGCAGGGTGACAAAG-3 NS-890: 5 -AGTAGA AACAAGGGTGTT-3 ) PCR, RNase A RNA PCR, DNAstar Seqman PCR ppoli-deons, 1.7 噬斑形成和免疫染色 deons R-PR8 0.3% BSA DMEM 10, MDCK 6, 37 o C 1 h,, (1 MEM, 1% agrose, 0.3% BSA, 100 μg/ml DEAE-Dextran, 1 μg/ml TPCK-Trypsin), 37 o C 5% CO 2 2 d 4 d,, 4% 15 min;, (0.1% TritonX-100, PBS) 20 min;, (0.5%, PBS) 1 h;, PBST(0.05% Tween20) 3, 1:100 A NP 37 o C 1 h; PBST 3, 1:2000 IgG 37 o C 1 h; PBST 3 AEC Chromogen, 10~20 min, 1.8 荧光实时定量 PCR 法检测病毒生长曲线 Youil [14], NP, ppoli-pr8np, PCR(qRT-PCR) MDCK : ppoli-pr8np, 10 ppoli-pr8np, NP (NP-884:5 -TGTATGGACCTGC CGTAGC-3 NP-1113:5 -CCCTCTTGGGAGCACC TT-3 ) qrt-pcr, : deons R-PR8 140 μl, Viral RNA Extraction Kit RNA, 60 μl RNase SYBR PrimeScrip RT-PCR Kit 2 qrt-pcr, deons R-PR8 RNA 8 μl Uni12 (2 μmol/l) 1 μl, 20 μl, 42 o C 1 h; deons R-PR8, NP-884 NP-1113, 20 μlpcr, MJ PCR, 3 : 95 o C, 60 o C, 70 o C, 40, (genome copies/ml) MDCK : (0.5 genome copy/cell) deons R-PR8 MDCK, 12 h~72 h 12 h, RNA qrt-pcr, 1.9 小鼠致病力实验 deons R-PR8 (10 4 PFU/ ) 6 BALB/c, 15, PBS, deons R-PR8,, ,, (10%, W/V), PBS 10, (0.2 ml/ ), 37 o C 48 h,, Reed-Muench EID 50, (logeid 50 /ml)

4 : A NS 结果 2.1 密码子去优化 NS1 基因的设计及分析 A [10],, NS1 151nt~480nt 68, 62%, 1 de/ each codon vs. usage table [15], A ( kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species= ) deoptimized NS, ( ) A, deons 病毒的拯救及鉴定 0.2 ml 12 9 SPF, 37 o C 48 h, 2 9,, Trizol RNA, RNA DNase I, RT-PCR NS, 900 bp, RNase A RNA ( 3),, DNA PCR, NS ppoli-deons NS, NS1 deons 2.3 噬斑形成实验, deons, MDCK 10 MDCK, 2 d 4 d,,,, 2 d, 40, R-PR8,, ( 4A); deons 2 d, R-PR8,, ( 4B), 4 d,, 4C 4D, R-PR8, 3~4 mm, deons, NS1, 2.4 MDCK 细胞中的病毒生长曲线的测定 deons, MDCK 10 图 1 NS1 基因 330 核苷酸 (151nt-480nt) 密码子去优化同义突变 Fig. 1 Synonymous codon-deoptimization in 330 nucleotides of NS1 gene (151 nt 480 nt). 1: the cdna sequence of PR8 NS1 gene(151 nt 480 nt, GenBank Accession No. AF389122); 2: the sequence with the deoptimized synonymous mutations (the synonymous mutations are shown in grey); 3: the deduced amino acids sequence(51 aa 160 aa).

5 724 ISSN CN /Q Chin J Biotech May 25, 2009 Vol.25 No.5 图 2 设计合成的 deoptimized NS 基因中密码子使用频率分析 Fig. 2 Codon usage analysis of synthetic deoptimized NS1 gene. Y axis: the relative codon frequency (RCF); X axis: amino acid and codon usage. The relative frequency of individual codon (RCF) is shown for each amino acid in black for influenza A virus genome and in white for the synthetic deoptimized NS1 gene. PPOLI-PR8NP qrt-pcr,, 3, R-PR8 deons (gene copies/ml) deons R-PR8 moi=0.5(gene copy/cell) MDCK, qrt-pcr, 24~72 h, deons R-PR8, 48 h,, deons 1000 ( 5) 2.5 小鼠致病力实验 10 4 PFU, R-PR8, ; deons,, ( 6) 3, deons R-PR8 100 ; 5, deons R-PR ; 7, deons, R-PR8, 1, deons PR8, 7,, deons 表 1 感染小鼠肺脏病毒滴定 Table 1 Virus titer in mice lungs after challenge with viruses Group Days post infection Virus titer (logeid 50 /ml±sd) deons ± ±1.4 7 ND (none detectable virus) R-PR ± ± ±0.6 3 讨论 图 3 救获病毒 NS 基因 RT-PCR 扩增 Fig. 3 RT-PCR amplification of the NS gene from the rescued virus. 1: DL 2000 marker; 2: negative control; 3: RT-PCR product of NS gene. 20, 2 2,

6 : A NS1 725 图 4 病毒噬斑形成实验 Fig. 4 Plague assay of R-PR8 and deons virus. (A, B) Microphotographs of the plaque with magnification of 40 times after 2 days of infection (A: R-PR8; B: deons). (C, D) Scanned pictures of the plaque after 4 days of infection (C: R-PR8; D: deons). 图 5 定量 PCR 测定病毒在 MDCK 细胞中的生长曲线 Fig. 5 Real-time quantitative PCR analysis of virus growth in MDCK cell., ; 3) mrna,,, [16,17] Mueller [18] Burns [19] (Poliovirus),,, CD155,,,,, NS1, NS1,, NS1 REFERENCES 图 6 小鼠体重变化曲线 Fig. 6 Body weight changes of mice after challenge with viruses. : 1) trna ; 2) mrna [1] Palese P, Garcia-Sastre A. Influenza vaccines: Present and future. J Clin Invest, 2002, 110(1): [2] Cox RJ, Brokstad KA, Ogra P. Influenza virus: Immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol, 2004, 59(1): [3] Palese P. Making better influenza virus vaccines? Emerg Infect Dis, 2006, 12(1): [4] Watanabe T, Watanabe S, Kida H, et al. Influenza A virus

7 726 ISSN CN /Q Chin J Biotech May 25, 2009 Vol.25 No.5 with defective M2 ion channel activity as a live vaccine. Virology, 2002, 299(2): [5] Watanabe T, Watanabe S, Kim JH, et al. Novel approach to the development of effective H5N1 influenza a virus vaccines: Use of M2 cytoplasmic tail mutants. J Virol, 2008, 82(5): [6] Talon J, Salvatore M, O Neill RE, et al. Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach. Proc Natl Acad Sci USA, 2000, 97(8): [7] Ferko B, Stasakova J, Romanova J, et al. Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes. J Virol, 2004, 78 (23): [8] Watanabe T, Watanabe S, Neumann G, et al. Immunogenicity and protective efficacy of replicationincompetent influenza virus-like particles. J Virol, 2002, 76(2): [9] Garcia-Sastre A, Egorov A, Matassov D, et al. Influenza A virus lacking the NS1 gene replicates in interferondeficient systems. Virology, 1998, 252(2): [10] Zhou T, Gu W, Ma J, et al. Analysis of synonymous codon usage in H5N1 virus and other influenza A viruses. Biosystems, 2005, 81(1): [11] Fujii K, Fujii Y, Noda T, et al. Importance of both the coding and the segment-specific noncoding regions of the influenza a virus NS segment for its efficient incorporation into virions. J Virol, 2005, 79(6): [12] Lamb RA, Choppin PW. Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proc Natl Acad Sci USA, 1979, 76(10): [13] Webster R, Cox N, Stohr K. WHO manual on animal influenza diagnosis and surveillance. 2 nd Ed. The World Health Organization(WHO), 2002: [14] Youil R, Su Q, Toner TJ, et al. Comparative study of influenza virus replication in Vero and MDCK cell lines. J Virol Methods, 2004, 120(1): [15] Fuhrmann M, Hausherr A, Ferbitz L, et al. Monitoring dynamic expression of nuclear genes in Chlamydomona reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol, 2004, 55(6): [16] Duan J, Wainwright MS, Comeron JM, et al. Synonymous mutations in the human dopamine receptor D2(DRD2) affect mrna stability and synthesis of the receptor. Hum Mol Genet, 2003, 12(3): [17] Duan J, Antezana MA. Mammalian mutation pressure, synonymous codon choice, and mrna degradation. J Mol Evol, 2003, 57(6): [18] Mueller S, Papamichail D, Coleman JR, et al. Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity. J Virol, 2006, 80(19): [19] Burns CC, Shaw J, Campagnoli R, et al. Modulation of poliovirus fitness in Hela cells by deoptimization of synonymous codon usage in the capsid region. J Virol, 2006, 80(7): 本期广告索引 GE Healthcare Promega Roche