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1 生物工程学报 Chin J Biotech 2009, October 25; 25(10): 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 16 L1 李巍巍 1,2, 何秀萍 1, 郭雪娜 1, 张振颖 1, 张博润 1, , : 为了实现人乳头瘤病毒 (Human papillomavirus, HPV)16 亚型衣壳蛋白 L1 在多形汉逊酵母 (Hansenula polymorpha) 中的高效表达, 根据 L1 蛋白的氨基酸序列及多形汉逊酵母的密码子偏爱性, 对 L1 蛋白的编码序列进行优化设计, 合成了完整的编码序列, 命名为 HPV16L1 以甲醇诱导型启动子 MOXp 和终止子 AOXTT 为表达调控元件, 以尿嘧啶合成相关基因 URA3 为筛选标记, 构建了 HPV16L1 的重组表达质粒 pymoxu-hpv16 用 Sac II 酶切质粒 pymoxu-hpv16 使其线性化, 电转化多形汉逊酵母菌株 H-ura3, 依据营养缺陷互补筛选重组菌株通过 PCR 扩增及 HPV16 L1 蛋白表达量分析表明已获得稳定高表达 L1 蛋白的重组汉逊酵母菌株 HP-U-16L 摇瓶发酵条件的初步优化表明, 以 YPM (ph 7.0) 为基础培养基进行诱导培养, 控制接种量使初始培养液 OD 600 为 1.0, 每隔 12 h 补加甲醇至终浓度为 1% (V/V), 37 o C 200 r/min 条件下诱导培养 72 h 后, HPV16 L1 蛋白的最高表达量为 78.6 mg/l 本研究为多形汉逊酵母源 HPV16 L1 疫苗的研制奠定了基础 : 人乳头瘤病毒 16 亚型, 衣壳蛋白 L1, 多形汉逊酵母, 优化表达 1 Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha Weiwei Li 1,2, Xiuping He 1, Xuena Guo 1, Zhenying Zhang 1, and Borun Zhang 1 1 Laboratory of Yeast Molecular Genetics and Breeding, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China 2 Graduate University of Chinese Academy of Sciences, Beijing , China Abstract: The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pmoxzα-a vector to generate plasmids pmoxz-hpv16n and pmoxz-hpv16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPV16L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (ph 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 C for 72 h, the recombinant produced 78.6 mg/l of L1 protein. This work offers the possibility for the production of prophylactic Received: July 6, 2009; Accepted: August 17, 2009 Corresponding author: Xiuping He. Tel: ; hexp@im.ac.cn

2 : 16 L vaccine for cervical carcinoma by H. polymorpha. Keywords: human papilomavirus, 16 L1 protein, Hansenula polymorpha, optimized expression (Cervical cancer), [1], , Zur Hausen (Human papillomavirus, HPV) DNA, HPV [2] (IARC) 22 (Invasive cervical cancer, ICC) HPV, ICC % HPV DNA, HPV HPV16 HPV18 [3] WHO 1992 HPV [4] HPV 14.2%, HPV16 HPV18 70% [5] HPV DNA, DNA, L1( 55 kd) (Virus-like particles, VLPs), VLPs, DNA [6] VLPs, [7-9], HPV16 L1 -, (GSK) - VLPs Merck VLPs, [10-11],,,,,,, [12],, B IFNa-2a [13-14] HPV16 L1, HPV16 L1, HPV16 L1, 1 材料和方法 1.1 材料 (Escherichia coli) DH5α [supe44 lacu169 (ϕ80lacz M15) hsdr17 reca1 enda1 gyra96 thi-1 rela1], (E. coli) TOP 10, (H. polymorpha) H-ura3, pbluscriptm13 YEp352 pmoxzα-a, T4 DNA Pfu Mix ( ) Zeocin Invitrogen HPV16L1 Santa Cruz (USA) PCR Western blotting 1.2 方法 HPV16L1 GenBank HPV16 L1 (GenBank Accession No. ACA14209),, L1, PCR, HPV16L1, 1.5 kb; DNA pbluscriptm13, pm13-hpv16l1,

3 1518 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No HPV16L1-L (5 -AGTAAGCTTATGT CTTTGTGGTTGCCATCT-3, Hind III ) HPV16L1-R (5 -GGCGCGGCCGC CTATTACAACTTTCTCTTCTT-3, Not I ), pm13-hpv16l1, PCR HPV16L1, PCR Hind III Not I, Hind III Not I pmoxzα-a, pmoxz- HPV16 MOXp-L (5 -CCTGAGCTCGTCG ACGCGGAG AACGATCTC-3, Sac I ) AOXt-R (5 -GCAGGATCCGCAAA TGGCATTCTGACATCC-3, BamH I ), pmoxz-hpv16, PCR 3.4 kb DNA, MOXp-HPV16L1- AOX1TT, Sac I BamH I PCR, YEp352, URA3 pymoxu-hpv Sac II pymoxu-hpv16, pymoxu-hpv16 (Bio-Rad Gene-Pulser Apparatus, 1.5 kv, 50 μf, 200 Ω, 3 ms) (H. polymorpha)h-ura3 H-ura3 (ura3), URA3, (SC) [15], PCR HPV16 L1 5 ml YPG (1%, 2%, 1% (V/V), ph), 37 o C 200 r/min 20 h 3000 g 5 min, 2, 50 ml YPM (1%, 2%, 1% (V/V) ) 250 ml, 37 o C 200 r/min, 12 h 0.5% 72 h,,, HPV16 L1, ph L g 5 min, (20 mmol/l NaH 2 PO 4, 2 mmol/l EDTA, 0.4 mol/l NaCl, NaOH ph 7.0) 1, 2 mmol/l PMSF, (400 W 10 s, 20 s, 80 ), g 10 min, 20 o C Bradford, [16] Western blotting L1, 12% SDS-PAGE [17], 150 ma 1 h, 1 h, HPV16L1 1 h,, HRP IgG 1 h,, HRP-DAB ( ) (Shimadzu dual-wavelength Tlc scanner cs-930, Japan) Western blotting SDS-PAGE 2 结果 2.1 密码子优化 HPV16L1 基因的合成和表达质粒的构建 DNAman HPV16 L1 HPV16L1-N (GenBank Accession No. EU430688), L1 ( interfaces/codonw.html), L1 GenBank L1 (GenBank Accession No. ACA14209),, GC, GC AT, HPV16 L1,

4 : 16 L PCR 1.5 kb L1 HPV16L1-N HPV16L1, pbluscriptm13, pm13- HPV16L1N pm13-hpv16l1, HPV16L1, (Met) (Phe) (Asp) (Glu) (Trp) HPV16L1-N,, ( 1) GC,, GC AT GenBank HPV16L1 pmoxzα-a Hind III Not I, pmoxz-hpv16 ( 2A) MOXp-L AOXt-R pmoxz-hpv kb DNA, MOXp AOX1TT HPV16L1 MOXp-HPV16L1- AOX1TT YEp352 Sac I BamH I, pymoxu- HPV16( 2B) pymoxu-hpv16 MOXp-HPV16L1-AOX1TT pymoxu-hpv16n (GenBank Accession No. GQ423063) RNAfold ( mrna ( 1), mrna, mrna pm13-hpv16l1, HPV16L1-L HPV16L1-R PCR, 表 1 优化后基因 HPV16L1 与天然基因 HPV16L1-N 的密码子使用对比 Table 1 Coden usage in HPV16L1 and HPV16L1-N Amino acids Coden usage in HPV16L1-N Coden usage in HPV16L1 Ala 6GCC+15GCA+9GCT 30GCT Arg 1CGG+4CGA+3CGC+11AGA 19AGA Asn 20AAT+8AAC 28AAC Cys 4TGC+8TGT 12TGT Gln 11CAA+9CAG 20CAA Gly 8GGA+14GGT+8GGC+4GGG 34GGT His 8CAT+2CAC 10CAC Ile 9ATA+1ATC+12ATT 7ATC+15ATT Leu 3CTT+5CTG+5TTG+7CTA+23TTA 43TTG Lys 18AAA+15AAG 33AAG Pro 16CCT+16CCA+5CCC 37CCA Ser 15TCT+5AGT+2AGC+5TCC+6TCA 27TCT+6TCC Thr 13ACT+1ACG+17ACA+11ACC 30ACT+12ACC Tyr 13TAT+9TAC 22TAC Val 5GTC+8GTA+14GTT+5GTG 26GTT+6GTC The number before the coden represents the times of coden usage. 图 1 HPV16 L1 蛋白天然 mrna 二级结构 (A) 和密码子优化后二级结构 (B) 比较 Fig. 1 Secondary structure of the native mrna (A) and the coden optimized mrna (B). 2.2 多形汉逊酵母重组菌株的构建与筛选 URA3 pymoxu-hpv16 pymoxu-hpv16n URA3 H-ura3, (SC), DNA,, PCR, 1.5 kb DNA, PCR, HPV16L1 HPV16L1-N

1520 ISSN1000-3061 CN11-1998/Q Chin J Biotech October 25

5 1520 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 图 2 Fig. 2 质粒 pmoxz-hpv16 (A) 和 pymoxu-hpv16 (B) 的物理图谱 Physical maps of plasmids pmoxz-hpv16 (A) and pymoxu-hpv16 (B). 2.3 HPV16 L1 蛋白的诱导表达和分析 37 o C 72 h,,, Western blotting, HPV16 L1, pymoxu-hpv16 HPV16 L1, 55 kd ( 3), HPV16 L1, HPV16L1-N, (Arg) CGG CGA 5, HPV16 L1,, HP-U-16L 2.4 HPV16 L1 诱导表达条件的优化, ph HPV16 L1 HP-U-16L YPG 20 h, 10% 50 ml YPM, 37 C (200 r/min), 12 h 24 h, 0.5% 1.0% 1.5% 72 h,, HPV16 L1 ( 4), 12 h 1.0%, L1, L1, 58.2 mg/l, L1, ;, [18-19] 图 3 HPV16 L1 蛋白表达的 Western blotting 分析 Fig. 3 Western blotting analysis of the expression of HPV16 L1 in H. polymorpha. M: protein marker; 1: negative control (protein sample of H. polymorpha H-ura3); 2 5: protein samples of transformants carrying the coden optimized HPV16L1 gene. HP-U-16L ph YPM ( 50 mmol/l ph 5.8~7.8 ), 12 h 1.0% 72 h, 5, ph, ph 6.2~6.6

6 : 16 L 图 4 甲醇添加方式对细胞生长和 HPV16 L1 表达的影响 Fig. 4 Effects of different methanol fed-method on the expression of HPV16 L1. Values are means of three replications ± standard deviation. : OD 600 ; : concentration of HPV16 L1. 图 5 培养液 ph 对细胞生长和 HPV16 L1 表达的影响 Fig. 5 Effects of ph on cell growth and the expression of HPV16 L1. Values are means of three replications ± standard deviation. : OD 600 ; : concentration of HPV16 L1. 图 6 初始接种量对细胞生长和 HPV16 L1 表达的影响 Fig. 6 Effects of inoculum size on cell growth and the expression of HPV16 L1. Values are means of three replications ± standard deviation. : OD 600 ; : concentration of HPV16 L1.,, L1 ph 7.0, 69.8 mg/l ph L1 50 ml YPM (ph 7.0), 12 h 1.0%, HPV16 L1,, L1 OD ( 6), L1 HP-U-16L YPG 20 h, YPM (ph 7.0), OD , 37 o C, 12 h 图 7 诱导时间对细胞生长和 HPV16 L1 表达的影响 Fig. 7 Time course of cell growth and HPV16 L1 production of recombinant H. polymorpha HP-U-16L in shake flask culture. Values are means of three replications ± standard deviation. : OD 600 ; : concentration of HPV16 L1. 1.0%, 12 h HPV16 L1, 96 h 7, 36 h, L1, 36 h, L1

7 1522 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10, 72 h, L1, L mg/l,, HPV16 L1, 72 h 3 讨论,,, 50, 70%,, (HPV),, HPV,, - [12-13], AT, HPV16L1 AT,,, HPV16 L1, HPV16 L1, Western blotting 55 kd,, HPV16 L1,,, ph HPV16 L1,,,,, HPV16 L1,, ph, ph 8.0, ph, 4.5~7.0, ph 7,, ph 7.0 HPV16 L1 20%, YPG ph 7.0 YPM, OD , 37 o C 200 r/min, 12 h 1%, 72 h, HPV16 L mg/l, 35% HPV, VLPs,, VLPs REFERENCES [1] Cornelison TL. Human papillomavirus genotype 16 vaccines for cervical cancer prophylaxis and treatment. Current Opinion Oncol, 2000, 12(5): [2] Gissmann L, zur Hausen H. Inverted repetitive sequences in human papilloma virus-1(hpv-1) DNA. Virology, 1977, 83(2): [3] Walboomer JM, Jacobs MV. Human papillomavirus is a necessary cause of invasive cervical cancer worldwise. J Pathol, 1999, 189 (1): [4] Dell G, Gaston K. Human papillomaviruses and their role in cervical cancer. Cell Mol Life Sci, 2001, 58(12/13): [5] Chen BL, Ma XD, Mu RH, et al. Induction of apoptosis in human cervical carcinoma cell line SiHa by antisense oligonucleotide of human papillomavirus type16 E6 gene. Fourth Mil Med Univ, 2000, 21(2): [6] Kirnbauer R, Booy F, Cheng N, et al. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci USA, 1992, 89(24): [7] Koutsky LA, Aμlt KA, Wheeler CM, et al. A controlled trial of a human papillomavirus type16 vaccine. N Engl J Med, 2002, 347(21): [8] Harper DM, Franco EL, Wheeler C, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of

8 : 16 L infection with human papillomavirus types 16 and 18 in young women: a randomized controlled trial. Lancet, 2004, 364(9447): [9] Villa LL, Costa RL, Petta CA, et al. Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomized double-blind lacebo-controlled multicentre Phase II efficacy trial. Lancet Oncol, 2005, 6(5): [10] Harper DM. Prophylactic human papillomavirus vaccines to prevent cervical cancer: review of the Phase II and III trials. Therapy, 2008, 5(3): [11] Stanley M. Human papillomavirus vaccines versus cervical cancer screening. Clin Onco, 2008, 20(6): [12] Gellissen G, Kunze G, Gaillardin C, et al. New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica a comparison. FEMS Yeast Res, 2005, 5(11): [13] Boer E, Steinborn G, Kunze G, et al. Yeast expression platforms. Appl Microbiol Biotechnol, 2007, 77(3): [14] Chen Z, Wang Z, He X, et al. Uricase production by a recombinant Hansenula polymorpha strain harbouring Candida utilis uricase gene. Appl Microbiol Biotechnol, 2008, 79(4): [15] Adams A, Gottschling DE, Kaiser CA, et al. Methods in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual. New York: Cold Spring Harbor Laboratory Press, [16] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(5): [17] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, [18] Ohashi R, Mochizuki E, Suzuki T. A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask. J Biosci Bioeng, 1999, 87(5): [19] Zhou XS, Fan WM, Zhang YX. Effects of different methanol feeding strategy on hirudin production in highdensity fermentation by recombinant Pichia pastoris. Chin J Biotech, 2002, 18(3): 周祥山, 范位民, 张元兴. 不同甲醇流加策略对重组毕赤酵母高密度发酵生产水蛭素的影响. 生物工程学报, 2002, 18(3): 我单位生物工程学报等 4 种期刊论文数据库制作完成并上网,,, 1, 4 1 1,,, , 1,,,,,, 2008, Open Access( ),,,, :

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i i ii 1 2 3 4 % N) 5 50.0 AMINO ACID #64 [modified by Administrator] IntAmp_1 nc 40.0 30.0 20.0 10.0 0.0-10.0 1 - ala - 10.483 2 - thr - 11.107 3 - gly - 11.872 4 - val - 13.458 5 - ser - 14.903 6 - pro