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 ppic9k-sm D1 用电穿孔法转化酵母菌 SMD1168, 在 MD 平板上筛选重组克隆, 用 G418 快速筛选高拷贝转化子, 阳性克隆经甲醇诱导表达后, 培养上清用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和免疫酶斑点法 (immunodot) 鉴定 结果显示 PCR 产物约为 360 bp, 符合预期, ppic9k-sm D1 重组阳性克隆双酶切鉴定正确, 测序结果与 GenBank 核酸数据库报道完全一致 表达产物 Sm D1 的分子量约 16 kd, 高拷贝毕赤酵母转化菌的表达水平明显高于低拷贝的 immunodot 证实表达产物具有天然 Sm D1 分子的免疫原性 阴性对照菌未见目的表达条带 Immunodot 的敏感性为 96%, 特异性为 100%, 与免疫印迹法 (immunoblot, IBT) 的符合率为 98% Sm D1 在巴斯德毕赤酵母中获得高效分泌表达, 为下一步研究打下了基础 关键词 : Sm D1 抗原, 克隆表达, 巴斯德毕赤酵母 Eukaryotic Expression and Primarily Application of Human Smith D1 Antigen in Methylotrophic Yeast Pichia pastoris YANG Xiang-Yue WU Wen-Bing LAN Xiao-Peng * (PLA Medical Labouratery Center, Fuzhou General Hospital, Fuzhou, Fujian 350025, China) Abstract: To clone, express and primarily use human autoantigen Sm D1 in methylotrophic yeast Pichia Pastoris. The gene Sm D1 was cloned by PCR.The PCR product was inserted into the vector ppic9k. The recombinant plasmid ppic9k- Sm D1 was transformed into yeast SMD1168 by electroporation. The positive clones were screened in MD plates. The high copy number transformants were rapidly selected by using G418 and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and immunodot. The PCR product was showed about 360 bp in size which was in accordance with predicted. The ppic9k-sm D1 showed the same seqencing result with GenBank s report and restriction enzyme analysis confirmed our prediction. The ppic9k-sm D1 positive clone produced an about 16 kd protein which had natural immunogenicity of human autoantigen Sm D1 by SDS-PAGE and immunodot. The sensitivity and specificity of immunodot were 96% and 100%, respectively. The agreement between immunodot and immunoblot was 98%. Successfully cloning and high-level expression of human autoantigen Sm D1 in methylotrophic yeast Pichia pastoris laid a foundation for further research work. Keywords: Sm D1 antigen, Cloning and expression, Pichia pastoris 基金项目 : (No. 2002J060) * 通讯作者 : : cyyh983@sina.com 收稿日期 :2008-05-28; 接受日期 :2008-09-11
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 assay, ELISA),,, Sm D1, (GST) Sm D1 [2],, (Pichia pastoris),,,, Sm D1 1 材料和方法 1.1 材料 JM109 SMD1168 (his4pep4) ppic9k, pgex-5t-sm D1 T4 DNA (PCR) DL2000 DNA TaKaRa DNA DNA G418 BSA YNB ENA ( ) DAB H 2 O 2 -Sm D1 Bio-Rad PE 2400 PCR ABI GSG-2000 LB(1% 0.5% 1% ), LBA(LB 100 μg/ml ), YEPD(1% 2% 2% 2% ), MD(1.34%YNB 4 10-5 % 2% 1.5% ), BMGY(1% 2% 1.34% YNB 1% 100 mmol/l ph6.0 4 10 5 % ), BMMY( BMGY 1% 0.5% ) 5 -CCTACGTAATGAAGCTCGT GAGATTTTTG-3 (TACGTA SnaB I ), 5 -ACGCGGCCGCTTATCGCCT AGGACCCCCTCT-3 (GCGGCCGC Not I ) 1.2 方法 1.2.1 PCR 扩增 Sm D1 基因 : pgex-5t- Sm D1 Sm D1, PCR 94 C 3 min; 94 C 45 s, 58 C 45 s, 72 C 45 s, 35 ; 72 C 10 min 10 μl 1.5%, 1.2.2 ppic9k-sm D1 的构建 : PCR ppic9k, SnaB I Not I,,, CaCl 2 E. coli JM109,, 1.2.3 ppic9k-sm D1 对毕赤酵母的转化及高拷贝阳性转化子的筛选 : Sal I ppic9k-sm D1, DNA (1300 V, 25 μf, 200 ) SMD1168, MD, MD 2 mg/ml G418 YEPD, 30 C 3 d, G418 3 mg/ml G418 YEPD, 30 C 3 d, 17 G418, 1.2.4 高拷贝转化子的 PCR 鉴定 : 17 DNA,, Sm D1 PCR, Sm D1 DNA 1.2.5 Sm D1 的诱导表达 : G418 1 100 ml BMGY, 30 C OD 600 3~ 6 5000 r/min, 5 min 25 ml
: SmD1 151 BMMY 30 C, 3 d, 0.5%,, 80%, 20 C ppic9k, 1.2.6 十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳 (SDS-PAGE): 20 C 20 μl 2, R250 1.2.7 Sm D1 蛋白的分离纯化 : SDS-PAGE Sm D1, ph 7.4 PBS 4 C, Sm D1-80 C 1.2.8 免疫酶斑点法 (immunodot) [3] : 1 μl 0.5 μl Sm D1 0.5 μl BSA, -Sm D1,, DAB H 2 O 2 1.2.9 immunodot 与免疫印迹法 (immunoblot, IBT) 的对比分析 : 50 IBT -Sm D1 SLE 50,, Sm D1 ( 2), ( 3) 图 2 重组质粒双酶切电泳图 Fig. 2 Restrictive digestion analysis of recombinant plasmid 1 2 3 4 M DNA marker. 1: Void plasmid before digestion; 2: Void plasmid after digestion; 3: Recombinant plasmid after digestion; 4: Recombinant plasmid before digestion; M: DNA marker(dl2000). 2 结果 2.1 人 Sm D1 抗原基因的扩增 PCR,, 250 bp 500 bp, (360 bp) ( 1) 图 1 人 Sm D1 抗原基因 PCR 产物电泳图 Fig. 1 Agarose gel electrophoresis of PCR product of Sm D1 M: DNA marker 1: PCR. M: DNA marker (DL2000) 1: PCR product of Sm D1. 2.2 重组质粒的鉴定, SnaB Not, 360 bp 图 3 重组质粒 5 端测序图 ( 划线处为 SnaBⅠ 酶切位点 ) Fig. 3 The 5 sequencing of recombinant vector (SnaB site is underlined) 2.3 高表达重组酵母菌的 PCR 鉴定 17 PCR,, 250 bp 500 bp, (360 bp) ( 4) 2.4 SDS-PAGE 分析 16 kd,,, ( 5) 2.5 immunodot 分析 BSA, 2, Sm D1, -Sm D1 ( 6)
152 微生物学通报 2009, Vol.36, No.1 3 讨论 图 4 高表达重组酵母菌 PCR 电泳图 Fig. 4 Agarose gel electrophoresis of PCR analyzing of recombinant yeast M: DNA marker; 1~17: PCR. M: DNA marker; 1~17: Recombinant yeast. 图 5 表达产物的 15% SDS-PAGE 图 Fig. 5 15% SDS-PAGE of the culture supernatant 1 2 3 4 ppic9k 5. 1: Expression product of low copy numbertransformant; 2: Expression product of high copy transformant; 3: Supernatant of high copy transformant before (NH 4 ) 2 SO 4 precipitation; 4: Negative control of ppic9k; 5: Protein marker. 图 6 表达产物的 immunodot 图 Fig. 6 Immunodot of the expression product 1: 1 µl ; 2: 0.5 μl ; 3: BSA. 1: Positive dot of 1 μl; 2: Positive dot of 0.5 μl; 3: BSA negative control. 2.6 两种方法检测抗 -Sm D1 抗体结果比较 50 IBT -Sm D1 SLE, immunodot 48 50, immunodot 96%(48/50), 100%(50/50), 98% (98/ 100), Sm D1 Sm D1,,,,,,,, ( ) ( ) ( ),, [4] [5] ppic9k,,, ppic9k G418 (kan gene), G418 [6], G418 ppic9k-sm D1, [7],, mrna [4],, (methanol utilization test, Mut) [8] Sm D1, Sm 9 (common core), Sm B/B Sm D, Sm B/B U1-RNP, (mixed connective tissue disease, MCTD) U1-RNP,, Sm D SLE Sm D 3 Sm D1 Sm D2 Sm D3, Sm D1 [1] Sm D1,, IBT (ENA ),,,
: SmD1 153,,,,, IBT,, IBT [9 11] Sm D1,, Sm D1,,, IBT, 参考文献 [1] Hoch SO, Elsenberg RA, Sharp GC Diverse antibody recognition patterns of the multiple Sm-D antigen polypeptides. Clin Immunol, 1999, 92(2): 203 208 [2],,. Smith D1., 2007, 30(6): 655 658. [3]..., 1992, pp.792 793. [4],,,.., 2000, 27: 371 373. [5],,,. cc1., 2004, 20: 241 243. [6] Scorer CA, Clare JJ, McCombie WR, et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology(N Y), 1994, 12: 181 184. [7] Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology(N Y), 1993, 11: 905 910. [8] Sreekrishna K, Brankamp RG, Kropp KE, et al.strategies for optimal synthosis and secretion of heterologous protein in the methylotrophic yeast Pichia pastoris. Gene, 1997, 190: 55 62. [9]. ENA., 1999, 3: 201 202. [10],,,. ENA., 2003, 3: 1423 1423. [11],.., 2005, 20: 26 27. 论文中计量单位的表示方法 稿件书写规范, GB3100-3102-93 ( ),, : d; h; min; s : mol/l, M ( ) N( ) : r/min, rpm : Pa kpa MPa : OD( ) : D kd, bp kb :,, : t(h) (, ) :, % : 20 cm 0.3 cm, 20 0.3 cm; 3 ~5 3~5 ; 3%~6% 3~6%