: A Hc SFV 125 toxin serotype A. Keywords: Semliki Forest virus, RNA replicon, botulinum neurotoxin serotype A, C-terminal fragment of heavy-chain rec

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劫炎靳
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1 生物工程学报 Chin J Biotech 2008, January 25; 24(1): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved. 研究简报 A Hc SFV 余云舟, 孙志伟, 王双, 俞炜源, : 为了在哺乳动物细胞中表达 A 型肉毒毒素 Hc 抗原, 构建了含 A 型肉毒毒素受体结合区 Hc 基因的基于 RNA 和 DNA 的重组 Semliki 森林病毒 (Semliki forest virus, SFV) 复制子表达载体 RNA 和 DNA 复制子载体转染 BHK21 细胞后, 经间接免疫荧光 Western 印迹和 ELISA 检测, 结果表明非分泌型和分泌型的 Hc 抗原在细胞中都得到了有效地表达 ; 而且复制子表达载体与辅助病毒载体共转染均可制备高滴度的重组病毒颗粒, 该重组病毒颗粒感染细胞后, 也都能表达 Hc 抗原以上结果表明, 基于 RNA 和 DNA 的重组 SFV 复制子表达载体在细胞中均可有效地表达 Hc 抗原和制备具有感染能力并能表达 Hc 抗原的重组病毒颗粒基于 RNA 和 DNA 的重组 SFV 复制子表达载体的构建和含 A 型肉毒毒素受体结合区 Hc 基因的重组病毒颗粒的获得, 为进一步观察 SFV 复制子疫苗的免疫原性奠定了基础, 从而为 A 型肉毒毒素新型疫苗的研制提供了新途经 : semliki 森林病毒, 复制子, A 型肉毒毒素, 受体结合区 Hc, 重组病毒颗粒 Expression of the Hc Fragment of Clostridium botulinum Neurotoxin Serotype A in Mammalian Cells with Recombinant Semliki Forest Virus Expression System Yunzhou Yu, Zhiwei Sun, Shuang Wang, and Weiyuan Yu Beijing Institute of Biotechnology, Beijing , China Abstract: To produce the C-terminal fragment of heavy-chain receptor (Hc) of the Clostridium botulinum neurotoxin serotype A (BoNT/A) in mammalian cells, we cloned the Hc gene from BoNT/A into the RNA and DNA-based Semliki Forest virus (SFV) replicon expression vectors, resulting in recombinant replicon expression vectors psmhc, psmshc, pscarhc, and pscarshc. Effective expression of non-secreted and secreted antigen Hc were confirmed by indirect imumofluorescence (IF), Western blot and ELISA after transfecting BHK21 cells with these replicon expression vectors based on both DNA and RNA. Recombinant virus particles (RVP) were prepared by cotranfecting these expression vectors with helper vectors (psfv-helper2 or pshcar) and the expression of antigen Hc was confirmed by indirect imumofluorescence (IF) and Western blot in cells (BHK21) that were infected with these recombinant virus particles (RVP). RNA and DNA-based recombinant SFV replicon expression vectors and recombinant virus particles (RVP) containing Hc gene all effectively expressed antigen fragment Hc in cells. This work laid a foundation for the study of the immunogenicity of SFV replicon vaccines and provided an alternative to develop new type vaccines against botulinum neuro- Received: March 26, 2007; Accepted: May 3, 2007 Corresponding author: Weiyuan Yu. Tel: ; yuwy@nic.bmi.ac.cn.

2 : A Hc SFV 125 toxin serotype A. Keywords: Semliki Forest virus, RNA replicon, botulinum neurotoxin serotype A, C-terminal fragment of heavy-chain receptor(hc), Recombinant virus particles (RVP) (Botulinum neurotoxin, BoNT),, (A~G), A [1], Hc,, [2] A Hc, Hc, A [3] Hc,,, A [4,5] F [6,7] DNA,,, A DNA Lee [8] (Venezuelan Equine Encephalitis Virus, VEEV, ) A, BoNT/A RNA/DNA,, [9,10], SFV semliki ( ),,,,, [11,12], A Hc SFV, DNA RNA, SFV 1 材料与方法 1.1 材料 BHK21 5%, 10 u/ml 10 mg/ml DMEM (Invitrogen) psm psms psfv-helper2 RNA SFV [13,14], pscar pshcar DNA SFV [15] pgemt-hc A Hc [3] 1.2 引物和寡核苷酸 Hc SFV : F-HB (BamH I) : 5 -cgcggatccgcaccatggaatacatcaagaacatcatcaatacctcc-3 ; F-HN (Nde I) : 5 -gccgcatatggaatacatcaagaacatcatcaatacctcc-3 ; R-HC(Cla I) : 5 -tgacatcgatttacagtggacgttcaccccaac PCR 和分子克隆 PCR, DNA, 1.4 体外转录体 RNA 的制备 Spe I, DNA (2 μg), Promega SP6 RNA, RNA [13] 1.5 RNA 和 DNA 转染细胞 RNA DNA Qiagen TransMessenger TM Transfection Reagent Invitrogen Lipofectamine TM 2000 [13,15] 1.6 间接免疫荧光 (IF) 和 Western 印迹检测 Hc 在细胞中表达 5% ( ) ; 1% BSA, ( ) Hc ( ), 37 1 h; PBS 3, 1% 1% BSA PBS

126 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25, 2008 Vol.24 No.1 1:200 FITC (IgG/FITC, Jackson Immuno Rearchlaboratories Inc.), 37 0.

3 126 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1 1:200 FITC (IgG/FITC, Jackson Immuno Rearchlaboratories Inc.), h; Passive Lysis Buffer (Promega) RVP,,, SDS-PAGE Western Santa Cruz Luminol reagent X 2 结果 2.1 含 Hc 基因的重组 SFV 复制子表达载体的构建 pgemt-hc, F-HB R-HC, PCR 1.3 kb Hc, BamH I Cla I, psm, psmhc; F-HN R-HC, PCR 1.3 kb Hc, Nde I Cla I, psms, psmshc Xba I Spe I RNA psmhc psmshc, Hc, pscar, DNA pscarhc pscarshc 2.2 Hc 基因在哺乳动物细胞中的表达 RNA Hc psmhc psmshc R-Hc R-SHc BHK21, A, IF 1a,, psm, Hc SFV RNA Western, 2, , Hc, Hc RNA DNA Hc DNA pscarshc pscarhc, Hc, 1 IF Hc RNA DNA SFV (400 ) Fig.1 Expression of Hc gene was detected in cells transfected with RNA and DNA-based replicon expression vectors by IF (a) 1: indicates cells transfected with RNA transcripts from psmhc; 2: indicates cells transfected with RNA transcripts from psmshc; 3: indicates cells transfected with RNA transcripts from psm (Negative control). (b) 4: indicates cells transfected with pscarhc; 5: indicates cells transfected with pscarshc; 6: indicates cells transfected with pscar (Negative control)

4 : A Hc SFV Hc Western blot Fig. 2 Expression of Hc was detected in the lysates from cells transfected with RNA transcripts from psmshc and psmhc by Western blot Lane 1: the lysates from cells transfected with RNA transcripts from psmhc; Lane 2: the lysates from cells transfected with RNA transcripts from psmshc; Lane 3: the lysates from cells transfected with RNA transcripts from psm (Negative control); Lane M: protein molecular marker. The arrow indicates the position of the Hc IF 1b,, pscar Hc SFV DNA Western, 3 RNA, DNA SFV Hc, S pscarshc Hc, 3(a-2) DNA DNA Hc, ELISA Hc DNA pscarshc A Hc, ( ), pscarshc Hc 2.3 重组病毒颗粒感染细胞及其在细胞中表达抗原基因 Hc ( RNA DNA), BHK21, IF, 4(1-2, 4-5),, 4(3, 6), 4 Hc, Western Hc ( ) IF, /ml,,,, SFV, Hc,, 3 讨论 3 Hc Western Fig. 3 Expression of Hc was detected in the lysates and the cell culture supernatant from cells transfected with pscarshc (a) and pscarhc (b) by Western blot 1: the lysates from cells transfected with pscarshc; 2: the cell culture supernatant from cells transfected with pscarshc; 3: the lysates from cells transfected with pscar (Negative control); 4: the lysates from cells transfected with pscarhc; 5: the cell culture supernatant from cells transfected with pscarhc; 6: the lysates from cells transfected with pscar (Negative control); M: protein molecular marker. The arrow indicates the position of the Hc, A,,,, (100 kd) (50 kd) L, N, (Hc),, [1], DNA,

128 ISSN1000-3061 CN11-1998/Q Chin J Biotech January 25, 2008 Vol.24 No.1 4 IF Hc (RVP) (400 ) Fig.

5 128 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1 4 IF Hc (RVP) (400 ) Fig.4 Expression of Hc gene was detected in cells infected with RVP contained Hc gene by IF (a) 1: indicates cells infected with RVP from co-transfected with RNA transcripts from psmhc and psfv-helper2; 2: indicates cells infected with RVP from co-transfected with RNA transcripts from psmshc and psfv-helper; 3: indicates cells infected with RVP from co-transfected with RNA transcripts from psm and psfv-helper2 (Negative control). (b) 4: indicates cells infected with RVP from co-transfected with pscarhc and pshcar; 5: indicates cells infected with RVP from co-transfected with pscarshc and pshcar; 6: indicates cells infected with RVP from co-transfected with pscar and pshcar (Negative control) [2] A Hc, [16,17] A Hc, Hc [3], SFV A Hc,, RNA DNA A Hc Hc SFV Hc,,, Th2,, [4,5] Hc SFV, REFERENCES [1] Turton K, Chaddock JA, Acharya KR. Botulinum and tetanus neurotoxtins: structure, function and therapeutic utility. Trends Biochem Sci, 2002, 27(11): [2] Middlebrook JL. Production of vaccines against leading biowarfare toxins can utilize DNA scientific technology. Advanced Drug Delivery Reviews, 2005, 57: [3] Yu YZ, Sun ZW, Wang S, et al. High-level expression of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen. Chinese Journal of Biotechnology, 2007, 23 (5): ,,,. A Hc., 2007, 23(5): [4] Jennifer Clayton, John L, Middlebrook. Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A. Vaccine, 2000, 18: [5] Yu RH, Shaio MF, Tang SS, et al. DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice. J Biomed Sci, 2000, 7: [6] Bennett AM, Perkins SD, Holley JL. DNA vaccination protects against botulinum neurotoxin type F. Vaccine, 2003, 21: [7] Jathoul AP, Holley JL, Garmory HS. Efficacy DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters. Vaccine, 2004, 22: [8] Lee JS, Pushko P, Parker MD, et al. Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system. Infect

6 : A Hc SFV 129 Immun, 2001, 69 (9): [9] Leitner WW, Hwang LN, deveer MJ, et al. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat Med, 2003, 9 (1): [10] Leitner WW, Hwang LN, Bergmann-Leitner ES, et al. Apoptosis is essential for the increased efficacy of alphavirus-based DNA vaccines. Vaccine, 2004, 20(11-12): [11] Leitner WW, Ying H, Restifo NP. DNA and RNA-based vaccines: principles, progress and prospects. Vaccine, 2000, 18: [12] Rayner JO, Dravga SA, Kamrud KI. Alphavirus vectors and vaccination. Rev Med Virol, 2002, 12(5): [13] Yu YZ, Sun ZW, Yu WY. Construction of DNA and RNA based on bifunctional replicon vector derived from Semliki Forest virus. Chinese Journal of Biotechnology, 2005, 21(5): ,,. DNA RNA semliki., 2005, 21(5): [14] Yu YZ, Sun ZW, Yu WY. Expression of domain 4 of the protective antigen from anthrax in mammalian cells using recombinant Semliki Forest virus replicon vectors. Bull Acad Mil Med Sci, 2006, 30(6): ,,. SFV., 2006, 30(6): [15] Yu YZ, Sun ZW, Liu ZG, et al. High-level expression of foreign genes in vivo and in vitro by improved DNA-based replicon vector derived from Semliki Forest virus. Prog Biochem Biophys, 2006, 33(1): ,,,. DNA Semliki., 2006, 33 (1): [16] Clayton MA, Clayton JM, Brown DR, et al. Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli. Infect Immun, 1995, 63: [17] Baldwin MR, Tepp WH, Pier CL, et al. Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E. Infect Immun, 2005, 73(10): 关于生物工程学报 2008 年度开始专刊申请的通知 cjb@im.ac.cn 3. cjb@im.ac.cn Tel:

10 Animal Husbandry & Veterinary Medicine 2012 Vol. 44 No. 4 cantly in the presence of indicating that the resistant protein was not expressed in the

10 Animal Husbandry & Veterinary Medicine 2012 Vol. 44 No. 4 cantly in the presence of indicating that the resistant protein was not expressed in the 2012 44 4 9 2 2 3 1. 225009 2. 200240 3. 200072 R PCR p215c3lyz R pqe-31 R IPTG Western blotting pqe- p215c3lyz COS- DH5α OD 600 Western blotting p215c3lyz Western blotting R R S852. 4 A 0529-5130 2012