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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 生物技术与方法 SBP 庞云, 龚立, 彭思扬, 朱乃硕, : 乙肝表面抗原结合蛋白 (HBsAg binding protein, SBP) 是本实验室发现的一种人源蛋白, 该蛋白与人乙型肝炎病毒 HBV 表面抗原 HBsAg 存在特异性的结合能力此前的研究证实 SBP 具有增强乙肝疫苗免疫效果的作用为进一步研究该蛋白的生理功能和作用机制, 利用毕赤酵母表达系统进行了 SBP 的表达菌株构建, 筛选得到了 SBP 的高效表达菌株发酵产物经过分离纯化, 最终得到了大量高纯度的真核来源的目的蛋白通过 SDS-PAGE 高效液相色谱 Western blotting 和质谱鉴定, 证实所得到的蛋白具有较高的纯度和完整性通过 ELISA 方法初步证实了其与乙肝表面抗原具有较好的结合能力该研究为进一步进行 SBP 的体内外功能研究及免疫增效研究打下了基础 : 毕赤酵母, 乙肝表面抗原结合蛋白, 表达与纯化 Expression, purification and characterization of HbsAg binding protein in Pichia pastoris Yun Pang, Li Gong, Siyang Peng, and Naishuo Zhu School of Life Science, Institutes of Biomedical Science, Fudan University, Shanghai , China Abstract: Human hepatitis B virus surface antigen (HBsAg) binding protein(sbp) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement. Keywords: Pichia pastoris, HBsAg binding protein, expression and purification Received: May 22, 2009; Accepted: July 17, 2009 Supported by: National Natural Science Foundation of China (Nos , ), National High Technology Research and Development Program of China (863 Program)(No. 2006AA02Z462). Corresponding author: Naishuo Zhu. Tel/ Fax: ; nszhu@sina.com 国家自然科学基金项目 (Nos , ), 国家高技术研究发展计划 (863 计划 )(No. 2006AA02Z462) 资助

2 : SBP 1565 (Hepatitis B, HB), 3.5 HBV [1],, 1.2 [2], [3],, 5%~10% [4], HBV, HBV [4], (Hepatitis B virus surface antigen, HBsAg), cdna 1 cdna cdna, 1 344,,, (HBV surface antigen binding protein, SBP, GenBank Access No. AY570731) [5], Western blotting ELISA SBP HBsAg ;, HBV-SBP HepG2 [5] ; SBP HBsAg, [6], SBP SBP,, SBP, SBP (Pichia pastoris),,,,,, [7],, SBP,, SBP 1 材料和方法 1.1 菌株和质粒 DH5α ; P. pastoris GS115, ppiczαa Invitrogen SBP pbv220-sbp 1.2 主要试剂 : LB(0.5% yeast extract, 1.0% Tryptone, 0.5% NaCl, 1.5% Agar, W/V), YPD(1% yeast extract, 2% peptone, 2% glucose, W/V), BMGY(1.18% KH 2 PO 4, 0.3% K 2 HPO 4 3H 2 O, 1.34% YNB, % biotin, 1% yeast extract, 2% peptone 2%, W/V), BMMY(1.18% KH 2 PO 4, 0.3% K 2 HPO 4 3H 2 O, 1.34% YNB, % biotin, 1% yeast extract, 2% peptone 0.5%, W/V) (Yeast extract) (Peptone) ; (YNB) BD ;, PCR, DNA Axygen ; T4 DNA, PrimeStar DNA, DL2000 DNA marker, marker, Xho I Not I TaKaRa ; Taq DNA dntp ; SBP [5] ; HRP IgG( ) ; Millipore ; ProteinA GE ; Zeocin Invitrogen ; PNGaseF NEB 1.3 方法 ppic-sbp pbv220-sbp, SBP-F (5 -CAG CTC GAG AAA AGA ATG GAC GTC TGG

3 1566 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 GGC CAA GG-3 ) SBP-R(5 -TGC GCG GCC GCT CAT CAT TTA CCC GGG GAC AGG GAG AG-3 ) SBP, Xho I Not I ( ) Xho I Not I, ppiczαa, Invitrogen, ppic-sbp ppic-sbp 20 ml Zeocin (25 μg/ml) LB, 500 ng/μl Dra I,, 1 μg/μl P. pastoris (Invitrogen) GS115, 80 μl 5~10 μg, : 1500 V, 200 Ω, 25 μf, 100 μg/ml Zeocin YPDS (YPD 1 mol/l sorbitol), 30 o C 1~2 d,, /,, SBP-F/R PCR, GS115 2 ml BMGY, 30 o C 200 r/min 24 h 50 ml BMGY, 30 o C 200 r/min 24 h (OD ), 15 ml BMMY,, 24 h 75 μl, 96 h, GS-pPIC SDS-PAGE 80~100 SDS-PAGE,,, GS-pPIC-SBP GS-pPIC-SBP,, : 0.5% 1.0% 1.5%, 2.0% 3.0% 4.0%; : 12 h 24 h 36 h 48 h 72 h 96 h 120 h; ph : ; EDTA : 0, 1 mmol/l 2 mmol/l 4 mmol/l 10 mmol/l; Casamino Acid ( ) GS-pPIC, SDS-PAGE, 10 μl,, Marker 1.0 SBP SBP 72 h,, 0.22 μm ; 10 kd 5~10 ; ph 7.0 PBS SBP, A (ProteinA) ELISA SBP ProteinA : ph 9.6 ProteinA 1 μg/ml, 100 μl, 3, 4 o C ; PBST 5 ; PBST 5%, 300 μl/, 37 o C 2 h; PBST 5 ; SBP, 100 μl; PBST 5 ; PBST 1:6000 SBP, 100 μl, 37 o C 2 h; PBST 5 ; PBST 1:5000 HRP-, 100 μl, 37 o C 1 h; PBST 5 ; 100 μl,, 0.5 h; 50 μl 2 mol/l, OD 492, SBP OD SBP ProteinA, ProteinA, : PBS (ph 7.0) ProteinA, 5 ml, 2.5 ml/min;, PBS (ph 7.0), 2.5 ml/min, UV280 ; ph mol/l

4 : SBP 1567, 1.0~2.0 ml/min,, 10:1 ph 9.0 Tris-Cl GS-pPIC-SBP GS h 19 ml, PEG6000 PEG :1, 4 o C, 1/5, Bradford, GS-pPIC-SBP GS115 5 Bradford,, : =GS-pPIC-SBP GS SBP Western blotting SBP 10 ng SDS-PAGE, 200 ma 2 h NC, SBP HRP IgG ( ) Western blotting SBP HPLC SBP 12 μl(2 mg/ml) C18 HPLC,,, SBP SBP 10 μg SDS-PAGE,,, (MALDI-TOF-TOF-MS) SBP HBsAg ELISA ph 9.6 HBsAg 1 μg/ml, 100 μl 3, 4 o C ; PBST 5 ; PBST 5%, 300 μl/, 37 o C 2 h; PBST 5 ; PBST SBP, mg/ml, 100 μl, 37 o C 2 h, 100 μl SBP ; PBST 5 ; PBST 1:4000 HRP IgG, 100 μl, 37 o C 1 h; PBST 5 ; 100 μl,, 0.5 h; 50 μl 2 mol/l, OD 492 ; SBP OD 2 结果 2.1 ppic-sbp 表达载体的构建及鉴定 pbv220-sbp PCR SBP ppiczαa,, ppic-sbp 2.2 毕赤酵母阳性转化子鉴定 SBP-F/R, PCR, pbv220-sbp, 1, SBP, SBP P. pastoris 2.3 重组 SBP 的表达检测 GS-pPIC-SBP GS h SDS-PAGE,, 2, GS-pPIC-SBP 38 kd, SBP, GS-pPIC, SBP, SBP 2.4 SBP 高表达菌株的筛选 96 h 10 μl SDS-PAGE,,,, ( 3) 80~100,, GS-pPIC-SBP 2.5 GS-pPIC-SBP 的表达条件优化分析 GS-pPIC, SBP

5 1568 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No.10 ( 4A), 1.0%~1.5%, 0.5% ; 2.0%, Kan Amp, ( 4B) EDTA ( 4C), EDTA,, 2 mmol/l, 图 2 SDS-PAGE 鉴定发酵上清中 SBP 的表达 Fig. 2 Identification of SBP expression by SDS-PAGE analysis. 1: protein marker; 2: supernatant of GS-pPIC-SBP; 3: supernatant of GS115, negative control. 图 1 PCR 鉴定毕赤酵母阳性转化子 Fig. 1 Identification of GS-pPIC-SBP by PCR. 1: DL2000 DNA marker; 2: negative control; 3: ppic-sbp transformants; 4: pbv220-sbp, positive control. 图 3 SDS-PAGE 法鉴定高表达菌株 Fig. 3 Identification of clone with high expression level. 1 8: SDS-PAGE of the supernatant of 8 strains, the expressing quantity of strains 4 and 5 are higher. 图 4 SDS-PAGE 分析确定蛋白表达最佳条件 Fig. 4 Optimization of SBP expressing conditions based on scan by SDS-PAGE and gel image analysis system. SDS-PAGE analysis of supernatant in different fermentation conditions. (A) Different MeOH concentrations(%). (B) Add(+) or not add(-) antibiotics (Kanamycin, Ampicillin). (C) Different EDTA concentrations (mmol/l). (D) ph of culture medium. (E) Time (hours) of fermentation. (F) Add (+) or not add(-) casamino acid.

6 : SBP 1569 ph ( 4D), ph (ph 6.0), ( 4E), 24 h,, 96 h Casamino Acid, ( 4F), 2.6 重组 SBP 具有与 ProteinA 结合的能力 ELISA SBP ProteinA, 5 GS115 ProteinA SBP ProteinA 图 6 Fig. 6 HPLC 法分析重组 SBP 纯度 Purity test of recombinant SBP by HPLC. 图 7 纯化后 SBP 的 SDS-PAGE 及 Western blotting 鉴定 Fig. 7 SDS-PAGE and Western blotting analysis of purified SBP. 1 L GS-pPIC-SBP mg/ml(bradford ) SBP 7.5 ml, 15.5 mg/l, 72.1% 图 5 重组 SBP 与 ProteinA 亲和能力的 ELISA 检测 Fig. 5 Examination of affinity between recombinant SBP and ProteinA. GS115: fermentation supernatant of GS115 strain, negative control; GS-pPIC-SBP: fermentation supernatant of SBP expressing strain. 2.7 SBP 的纯化和纯度分析 GS-pPIC-SBP ProteinA, HPLC 95% ( 6) Western blotting, SBP SBP( 7) 2.8 目的蛋白表达量的定量分析 GS-pPIC-SBP GS115, 1.3.6, SBP 21.5 mg/l 图 8 Bradford 法测定 GS-pPIC-SBP 及 GS-pPIC 诱导上清中总蛋白浓度 Fig. 8 Total protein concentration of supernatants after inducement.

7 1570 ISSN CN /Q Chin J Biotech October 25, 2009 Vol.25 No 目的蛋白 SBP 具有与 HBsAg 的结合能力 ELISA SBP HBsAg ( 9), SBP HBsAg, 图 9 ELISA 法测定重组 SBP 与 HBsAg 的结合能力 Fig. 9 Examination of affinity between recombinant SBP and HBsAg by ELISA. 3 讨论 SBP, P. pastoris GS115, 21.5 mg/l, 95% SBP, 15.5 mg/l, SBP,, SBP (Pichia pastoris) :, ;,,,, ; P. pastoris,, ;,, [6-8], P. pastoris [9], [10-11] P. pastoris,,, :, 72~96 h, 30 o C,, P. pastoris, YAP3 P. pastoris, thsa P. pastoris [12], SBP SBP,,,,, SBP,, SBP,, REFERENCES [1] Lavanchy D. Worldwide epidemiology of HBV infection, disease burden, and vaccine prevention. J Clin Virol, 2005, 34(Suppl.1): S1 S3. [2] Parkin DM, Pisani P, Munoz N, et al. The global health burden of infection associated cancers. Cancer Surv, 1999, 33: [3] The world health report 2002: reducing risks, promoting healthy life. Geneva: World Health Organization, 2002: [4] Sophie O, Xuan N, Julien B. Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and

8 : SBP 1571 purification for in vitro diagnosis. Protein Expr Purif, 2007, 56(2): [5] Chen YY, Zhu NS. Screening, expression and characterization of a novel protein binding to Hepatitis B surface antigen. Chin J Biochem Mol Biol, 2005, 21(1): 陈媛媛, 朱乃硕. 一种新的乙型肝炎病毒表面抗原结合蛋白的筛选, 表达及其生物学活性的初步研究. 中国生物化学与分子生物学报, 2005, 21(1): [6] Li HQ, Zhu NS. Preparation of the HBV Surface Antigen Binding Protein and the functional study. J Fudan Univ Natur Sci, 2007, 46(3): 李华青, 朱乃硕. 重组乙肝表面抗原结合蛋白 (SBP) 的制备及功能研究. 复旦学报 ( 自然科学版 ), 2007, 46(3): [7] Dou WF, Lei JY, Zhang LF et al. Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagons-like-1 peptide mutants in Pichia pastoris. Protein Expr Purif, 2008, 61(1): [8] Anup KU, Da le EE. Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris. Protein Expr Purif, 2008, 59(2): [9] Cereghino JL, Cregg JM, Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbil Rev, 2000, 24(1): [10] Vervecken W, Kaigorodov V, Callewaert N, et al. In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microbiol, 2004, 70: [11] Chiba Y, Suzuki M, Yoshida S, et al. Production of human compatible high mannose-type sugar chains in Saccharomyces cerevisiae. J Biol Chem, 1998, 273(41): [12] Chen Y, Yuan HY, Li H, et al. Reduction of rhsa degradation during fermentation in Pichia pastoris by disrupting YPS1 gene. J Fudan Univ Natur Sci, 2008, 47(3): 陈莹, 袁汉英, 李海, 等. 毕赤酵母 YPS1 基因的失活对减少重组人血清白蛋白降解的作用. 复旦学报 ( 自然科学版 ), 2008, 47(3): 科学出版社科学出版中心生命科学分社新书推介张阳德主编年 9 月出版 ; ; ; ;, ; ;, ;,

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂中国细胞生物学学报 Chinese Journal of Cell Biology 2011, 33(4): 379-384 http://www.cjcb.org 全小麦啤酒酵母菌株的诱变育种及应用研究袁仲 1* 张百胜 1 张慎举 1 马绮云 2 陈柯羽 2 ( 1 商丘职业技术学院园林食品加工系,