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1 生物工程学报 Chin J Biotech 2008, March 25; 24(3): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved 研究报告杨欢利, 赵西彪, 宋晓娟, 丁家桐, : 为了获得长效 FSH 制剂, 利用重叠 PCR 技术将山羊 FSH 的 α, β 亚单位, 通过 hcgβ 亚单位羧基末端延长肽 (CTP) 基因序列的连接, 构建成单链长效类似物基因 FSHβ-CTP-α 将其克隆至表达载体 ppic9k, 重组载体线性化处理后电转化至毕赤酵母 GS115 中, 经判型和 G418 筛选后获得高拷贝菌株 His + Mut + 经甲醇诱导表达后, 进行 SDS-PAGE 和 Western blot 分析表明 : 重组 FSHβ-CTP-α 的转化子可以正确有效地表达目的蛋白, 分子量约为 29 kd 放射免疫分析法 (RIA) 测定表达上清, 高拷贝转化子的平均表达量为 miu/ml, 显著高于低拷贝转化子的平均 miu/ml 的表达量, 为 FSH 的结构研究和长效 FSH 制剂的生产奠定了基础 : 山羊促卵泡素, 长效类似物基因, 毕赤酵母 Expression of Goat Follicle-stimulating Hormone Analogous Gene in Pichia pastoris Huanli Yang, Xibiao Zhao, Xiaojuan Song, and Jiatong Ding College of Animal Science and Technology, Yangzhou University, Yangzhou , China Abstract: In order to obtain the long-acting FSH preparation, the single strand long-acting analogous gene FSHβ-CTP-α was successfully constructed by the C-terminal peptide(ctp) of carboxyl-terminal region of human chorionic gonadotropin with the goat FSHα-subunit and β-subunit genes, then it was inserted into ppic9k vector.the recombinant plasmid ppic9k FSHβ-CTP-α was transformed into Pichia pastoris GS115 by electroporation. The multi-copy inserts His + Mut + were gained by the screening of phenotype and hyper-resistance to G418.After methanol induction, the supernatant was analysised by SDS-Polyacrylamide Gen Electrophoresis and Western blot. The results show that the transformants of FSHβ-CTP-α could express the objective protein successfully and the molecular weight is about 29 kd. The concentration of supernatant was detected by Radio-immunoassay and the average expression of multi-inserts is miu/ml and the low-inserts is miu/ml. The expression of multi-inserts is higher than the low-inserts significantly. This research lay the foundation for studying the structure of FSH and the production of long-acting FSH preparation. Keywords: follicular-stimulating hormone, long-acting analogous gene, Pichia pastoris (FSH), (LH) (TSH) (CG), α β, α, β, Received: June 26, 2007; Accepted: August 21, 2007 Supported by: the Natural Science Foundation of Jiangsu Province(No. BK ). Corresponding author: Jiatong Ding. Tel: ; Fax: ; jtding@yzu.edu.cn 江苏省自然科学基金项目资助 (No. BK )

2 410 ISSN CN /Q Chin J Biotech March 25, 2008 Vol.24 No.3 [1] FSH,,, [2,3] FSH,, [4] FSH, FSH,,, [5,6], ; FSH [7 9], FSH 2~2.5 h, (hcg) 12~36 h [10], hcgβ (CTP), hcg [11] Fares [12] FSH,, ;,, [4] FSH FSHβ-CTP-α ppic9k, GS115, G418 His + Mut +,, FSH 1 材料和方法 1.1 材料 pvitrofshβ-ctp-α DH5α ; ppic9k GS EcoR I Not I Sal I, Pyrobest TM DNA Polymerase, DNA Ligation Kit Ver 2.0, MiniBEST Plasmid Purification Kit Ver 2.0, DL2 000, DL15 000, Agarose Gel DNA Purification Kit Ver 2.0 TaKaRa DNA, YNB, G418, -FSHβ IgG-HRP, FSH LB : 5 g/l, 10 g/l, NaCl 10 g/l; YPD BMGY BMMY MD MM [13] PCR ppic9k FSHβ-CTP-α cdna, FSHβ-CTP-α F1 F2 5 AOX1 3 AOX1, : F1 5 ATAGAATTCAGAAGCTGCGAGCTGA CC3 ( EcoR I ) F2 5 TATGCGGCCGCTTAAGATTTGTGATAA TAA3 ( Not I ) 5 AOX1 5 -GACTGGTTCCAATTGACAAGC-3 3 AOX1 5 -GCAAATGGCATTCTGACATCC 方法 FSH PCR FSHα FSHβ hcg (CTP), FSH FSHβ-CTP-α, [14] ppic9kfshβ-ctp-α pvitrofshβ- CTP-α, F1 F2 PCR, PCR : 94 o C 1 min; 94 o C 45 s, 58.7 o C 30 s, 72 o C 1 min, 30 ;, 72 o C 2 min PCR EcoR I Not I, Gene Tools, DNA Ligation Kit Ver o C 30 min DH5α,

3 : 411, EcoR I Not I PCR, GS115 ppic9kfshβ- CTP-α Sal I,, 0.5~1 µg/µl, GS115, 1500 V, 5 ms, Electroporator 2510, 30 2~3 d MD G418 YPD, G mg/ml 0.75 mg/ml 1.00 mg/ml 1.50 mg/ml 2.00 mg/ml 2.5 mg/ml 3.00 mg/ml, Invitrogen Multi- Cop Pichia Expression Kit PCR G418 MD, 30 2 d; MD MM MD, 30 2 d; MM MD,, His + Mut + His + Mut + YPD, [15]; 5 AOX1 3 AOX1 PCR, PCR : 94 4 min; 94 1 min, min, 72 1 min, 30 ;, 72 o C 2 min His + Mut + 25 ml BMGY 250 ml, r/min OD 600 2~6, 4500 r/min 8 min ; 100 ml BMMY 1 L, r/min ; 24 h 1 ml, 100% 0.5%, 5 min, SDS-PAGE Western blot 80,, SDS min, SDS-PAGE, 5% 15%,, Westren blot, [16] , FSH I 125 -FSH, 16~18 h, 4000 r/min 25 min (CPM), CPM, CPM 2 结果 2.1 FSHβ-CTP-α 基因的 PCR 扩增 FSHβ-CTP-α 782 bp, PCR 1.5% 图 1 FSHβ-CTP-α 的 PCR 扩增 Fig. 1 The product of FSHβ-CTP-α by PCR M: DNA marker DL2 000; 1~2: PCR products 2.2 重组表达载体 ppic9kfshβ-ctp-a 的酶切鉴定 ppic9k 9.3 kb, 782 bp, EcoR I Not I : 9.3 kb, 775 bp;, FSHβ-CTP-α 2.3 MD,

4 412 ISSN CN /Q Chin J Biotech March 25, 2008 Vol.24 No.3 G418 YPD, 4 mg/ml G418 YPD 6, 0.5 mg/ml G418 YPD 29 kd, 48 h, 72 h 168 h Western blot 29 kd FSHβ-CTP-α 图 4 SDS-PAGE 分析 FSHβ-CTP-α 基因不同时间的表达 Fig. 4 SDS-PAGE analysis of the expression of FSHβ-CTP- α at different times M: low molecular weight standard protein marker; 1~8: the objective protein expressed at different times 图 2 Fig. 2 重组载体 ppic9kfshβ-ctp-α 的酶切鉴定 Identification of ppic9kfshβ-ctp-α digested by EcoR I and Not I M: DNA marker DL PCR His + Mut +, 5 AOX1 3 AOX1 PCR, : 2.2 kb, 1267 bp; 2.2 kb (AOX1), 1267 bp 775 bp AOX1 492 bp, 图 5 FSHβ-CTP-α 基因表达蛋白的 Western blot 分析 Fig. 5 Western blot analysis of the protein expressed in Pichia pastoris M: protein marker; 1,2: identification of objective protein by Western blot 2.6 目的蛋白的放射免疫检测 (RIA), SPSS11.0, 6 图 3 阳性重组子的 PCR 鉴定 Fig. 3 Identification of positive recombinants by PCR M: DNA marker DL15 000; 1~3: the products of positive recombinants amplified by PCR 2.5 诱导表达产物的 SDS-PAGE 和 Western blot 分析 SDS-PAGE, Marker 图 6 FSHβ-CTP-α 基因表达蛋白的放射免疫检测 Fig. 6 Determination of objective protein by RIA, FSHβ-CTP-α

5 : 413, CV 10%, CV 15% 3 讨论 PCR,, [17] FSH 2, [18] Sugahara [19] FSH C N FSH CHO CTP 29 4 O-, Matzuk [20], CTP hcg, PCR FSHβ-CTP-α,, CTP FSH FSH 2,, α 52/78, β 7/24,, FSH ( ),, α-, Mitali samaddar [9] FSHβ FSHβ α-,, FSHβ α-, FSHβ, 230 ng/ml 4 μg/ml, PCR, α-, ( ),, Clare [21],,, 1~8, (HbsAg) [22], [23], mrna G418 YPD, 4 mg/ml G418 6,,, Southern blot, 20%~30% [24], FSH 16% [25] 29 kd, FSHβ-CTP-α kd, FSH,, ; FSH, REFERENCES [1] Ulloa-Aguirre A,Timossi C.Biochemical and functional aspects of gonadotropin-releasing hormone and gonadotrophins. Reproductive Biomedicine Online, 2000, 1(2): [2] Zhang ZC. The Reproduction of Livestock (3rd ed). Peking: Publishing Company of Chinese Agriculture, 2000, 41.. ( ). :, 2000, 41. [3] Rose MP, Gaines Das RE, Balen AA. Definition and measurement of follicle stimulating hormone. Endocrine Reviews, 2000, 21(1): [4] Rupali Gadkari,Rahul Deshpande, Rajan R.Dighe. Hyperexpression and purification of biologically active human luteinizing hormone and human chorionic gonadotropin using the methylotropic yeast Pichia pastoris. Protein Expression & Purification, 2003, 32: [5] Lasmezas CI, Deslys JP, Demainay R, et al. BSE transmission to macaqnes. Nature, 1996, 381: [6] Zhou JM. Prion diseases and the protein only hypothesis.

6 414 ISSN CN /Q Chin J Biotech March 25, 2008 Vol.24 No.3 Prog Bichem Biophys, 2004, 31(2): Prion proteinonly., 2004, 31(2): [7] Kobayashi M, Morita T, Ikeguchi K, et al. In vivo biological activity of recombinant goldfish gonadotropins produced by baculovirus in silk worm larvae. Aquaculture, 2006, 256: [8] Kumar TR, Schuff KG, Nusser KD, et al. Gonadotrophspecific expression of the human follicle stimulating hormone β gene in transgenic mice. Molecular and Cellular Endocrinology, 2006, 247: [9] Samaddar M, Catterall JF, Dighe RR. Expression of biologically active ß subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris. Protein Expression & Purification, 1997, 10: [10] Wang YF. Reproductive Medicine. Peking: Publishing Company of People s sanitation, 1991, :, 1991, 86. [11] Vicenta GC, Irving B. Novel recombinant gonadotropins. Trends in Endocrinology and Metabolism, 2001, 12(2): 72. [12] Nickle GM, Becker MD, Nicholas S, et al. First live birth after ovarian stimulation using a chimeric long-acting human recombinant follicle-stimulating hormone (FSH) agonist (recfsh-ctp) for in vitro fertilization. Fertility and Sterility, 2003, 79(3): [13] Invitrogen.Multi-copy Pichia Expression Kit, Version D, [14] Sun XP, Yan ZJ, Yang HL, et al. Construction and expression of goat follicle-stimulating hormone analogous gene. Acta Veterinaria Zootechnica Sinica, 2006, 37(12): ,,,. FSH., 2006, 37(12): [15] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A laboratory Manual 2 nd ed. New York: Cold Spring Harbor Laboratory Press, [16] Wang TH, Feng ZT, Jean Philippe Merlio. Theory and Technology of Molecular Hybridize. Peking: Publishing Company of Science, 2005, ,.. :, 2005, [17] Zhang M, Si LS. Recombinant PCR-the technology of rapid recombine gene in vitro. Journal of Xi an Medical University, 2001, 22(3): ,. PCR., 2001, 22(3): [18] Samaddar M, Babu PS, Catterall JF. Identification of an attenuating region in the bovine follicle-stimulating hormone beta subunit mrna that decrease its expression in E. coli. Gene, 1999, 228(1-2): [19] Sugahara T, Grootenhuis PD, Sato A, et al. Expression of biologically active genes encoding the common alpha subunit and either the CG beta of FSH beta subunit: role of linkersequence. Mol Cell Endocrinol, 1996, 125(1-2): [20] Matzuk MM, Boime I. The role of the asparagines-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin. Cell Biol, 1988, 106: [21] Clare JJ, Rayment FB, Ballantine SP, et al. High-level expression of tetanus toxion fragment C in Pichia pastoris containing multiple tanden integrations of gene. Biotechnology (NY), 1991, 9: 445. [22] Vassileva A, Chugh DA, Swaminathan S, et al. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression & Purification, 2001, 21: [23] Li HZ, Li LZ, Sun QM, et al. Strategies for optimizing Pichia pastoris expression system. Acta Microbiologica Sinica, 2003, 43(2).,,,.., 2003, 43(2): [24] Muyan M, Boine I. The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin subunit effects on the processing of the Asn-linked oligosaccharides. Mol Endocrinol, 1998, 12: 766. [25] Dong W. Reproductive Hormone of Livestock. Peking: Publishing Company of Agriculture, 1983, :, 1983, 本期广告索引 GE Healthcare Roche ( ) / Promega ( )

150 微生物学通报 2009, Vol.36, No.1 (systemic lupus erythematosus, SLE), Sm(B B D1), Sm D1, SLE [1], Sm D1 (immunoblot, IBT) (enzyme linked immunosorbent as

150 微生物学通报 2009, Vol.36, No.1 (systemic lupus erythematosus, SLE), Sm(B B D1), Sm D1, SLE [1], Sm D1 (immunoblot, IBT) (enzyme linked immunosorbent as 微生物学通报 JAN 20, 2009, 36(1): 149~153 Microbiology tongbao@im.ac.cn 2009 by Institute of Microbiology, CAS 生物实验室人 SmD1 抗原的酵母真核表达及其初步应用 * 杨湘越吴文冰兰小鹏 ( 350025) 摘要 : 通过 PCR 扩增 Sm D1 基因, 与酵母表达载体 ppic9k 重组, 构建表达质粒