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1 重组七鳃鳗 PHB2 蛋白的原核表达及纯化李铁松, 王颖, 高杨, 李庆伟 (, ) 摘要 : 为了研究 PHB2 蛋白的生物学活性及后续抗体的制备, 作者以前期构建的 pmd18-t-lm-phb2 质粒为模板, PCR 扩增 Lm-PHB2 基因全长 CDS 区, PCR 产物经 Hind III 和 EcoR I 双酶切后连接至表达载体 pet-32a, 构建重组质粒 pet-32a-lm-phb2 将鉴定正确的重组质粒转化入大肠杆菌(Escherichia coli)rosetta Blue, 经 IPTG 诱导表达后, 表达产物通过 SDS-PAGE 和 Western blotting 进行检测, 利用镍柱亲和层析纯化得到纯度较高的目的蛋白 rlm-phb2 结果表明, 经 PCR 双酶切和测序鉴定, 所构建的重组质粒 pet-32a-lm-phb2 序列正确, 表达产物存在于裂解菌液的上清和沉淀中, 经 SDS-PAGE 和 Western blotting 证实在约 47 ku 处有目的蛋白 rlm-phb2 的表达条带本研究构建了重组七鳃鳗 (Lampetra japonica)phb2 蛋白的原核表达载体, 并大量表达和纯化目的蛋白, 为该蛋白后续的抗体制备及生物活性研究奠定了基础关键词 : 海洋生物学 ; 七鳃鳗 (Lampetra japonica); PHB2; 原核表达 ; 蛋白纯化中图分类号 : R915 文献标识码 : A 文章编号 : (2015) doi: /hykx (Lampetra japonica),,,, Prohibitin(PHB), [1] PHB PHB1 PHB2,, PHB [2-5], DNA [2, 6],, PHB,,, [7-11] PHB, PHB2 (, reactive oxygen species, ROS) [12] PHB2,, [13] ROS,, PHB PHB2,,, T [12] PHB2 PHB, PHB1, [13] PHB2, PHB2, pmd18-t-lm-phb2, Lm-PHB2 His pet-32a,, : ; : : ( ); (2010J21DW018) : (1976-),,,,,, sally_ts_li@163.com;,,, liqw@263.net Marine Sciences / Vol. 39, No. 4 /

2 1 材料和方法 1.1 菌株和质粒 pet-32a Rosetta Blue, Rosetta Blue, pmd18-t-lm-phb2 1.2 试剂和药品 Taq Hind III EcoR I T4-DNA Ligase DNA Marker DNA TaKaRa ; ; His Bind Column IPTG PVDF Anti-PHB2 ; Marker ;,,,,,, Tris, SDS,,,,, Tween 20,,,, IgG(H+L) 1.3 引物的设计及合成 Ensembl PHB2, pet-32a, primer 6.0 PCR Hind III, EcoR I, : Forward( ): 5'- GGAATTCCATGGCT- CAGCAGCTCAAGGA-3 Reverse( ): 5 - CCCAAGCTTGGGC- TTCTTTTTCACCGAC 目的基因的扩增 pmd18-t-lm-phb2, Lm-PHB2 (CDS ) PCR : 94 5 min; s, s, 72 1 min, 30 ; min PCR 1%, DNA 1.5 表达载体的构建 PCR pet-32a Hind III EcoR I, PCR DNA, 16 6 h, Rosetta Blue,, PCR, 1.6 重组蛋白的诱导表达及纯化 50 mg/l (Amp) LB, 37 (12~16 h), ml LB ( 50 mg/l Amp), 37 OD ~0.6, 1.0 mmol/l IPTG, 37 5 h, rlm-phb2 12%SDS-PAGE, Western blotting SDS-PAGE PVDF, Marker, PVDF ( 5% TBST) 37, PHB2 ( ) IgG(H+L) ( ), ECL His Bind Column,, SDS-PAGE rlm- PHB2 2 实验结果 2.1 Lm-PHB2 基因的扩增 Lm-PHB2 CDS, PCR 1%, PCR 900 bp, Lm-PHB2 CDS ( 1), PCR Lm-PHB2 CDS 1 Lm-PHB2 CDS PCR Fig. 1 PCR products of Lm-PHB2 gene CDS region M.DNA Marker DL2000;1.PCR M.DNA Marker DL2000;1.PCR products 38 / 2015 / 39 / 4

2.2 重组质粒 pet32a-lm-phb2 的鉴定 2.2.1 PCR Lm-PHB2 CDS PCR, pet-32a, Rosetta blue,, 1~3, PCR, 900 bp ( 2), Lm-PHB2 CDS pet-32a 3 EcoR I Hind III Fig.

3 2.2 重组质粒 pet32a-lm-phb2 的鉴定 PCR Lm-PHB2 CDS PCR, pet-32a, Rosetta blue,, 1~3, PCR, 900 bp ( 2), Lm-PHB2 CDS pet-32a 3 EcoR I Hind III Fig. 3 Identification of pet-32a-lm-phb2 by restriction enzyme digestion M1.Marker DL2000; M2.λ-EcoT14 I digest DNA Marker; 1.pET-32a- Lm-PHB2; 2.pET-32a-Lm-PHB2/Hind III+EcoR I 2 pet32a-lm-phb2 PCR Fig. 2 Identification of pet32a-lm-phb2 by PCR M.Marker DL2000;1.pET32a-Lm-PHB2 M.Marker DL2000;1.Positive clone with pet32a-lm-phb PCR,, Hind III EcoR I ( 3),,, 900 bp, Lm-PHB2 CDS ; 5900 bp, pet-32a, PCR pet-32a-lm- PHB2, Ensembl PHB2, Lm-PHB2,, 2.3 重组蛋白 rlm-phb2 的表达及纯化 rlm-phb2 SDS-PAGE IPTG, SDS-PAGE, 47 ku ( 4, ),, rlm-phb2 4 SDS-PAGE rlm-phb2 Rosetta Blue Fig. 4 Expression of rlm-phb2 in Rosetta Blue tested by SDS-PAGE M. Protein Marker; 1. ; 2. ; 3, 4. IPTG Rosetta blue/pet-32a-lm-phb2 ;5. IPTG Rosetta blue/pet-32a-lm-phb2 ;6. IPTG Rosetta blue/pet-32a-lm-phb2 M. Protein Marker; 1. Supernatant of uninduced control; 2. Deposit of uninduced control; 3, 4..Bacteria liquid of Rosetta blue/pet-32a-lm-phb2 induced by IPTG; 5. Supernatant of Rosetta blue/pet-32a-lm-phb2 induced by IPTG; 6. Deposit of Rosetta blue/pet-32a-lm-phb2 induced by IPTG rlm-phb2 rlm-phb2 His Bind Column, SDS-PAGE, 47 ku ( 5),, rlm-phb2, 210 mg/l 2.4 重组蛋白 rlm-phb2 的 Western Blotting 分析 PHB2 (1: 1000) Western blotting, ( 6), Marine Sciences / Vol. 39, No. 4 /

Fig. 5 5 rlm-phb2 Elution and purification of rlm-phb2 by His system A. rlm-phb2 ;M.protein marker;1-4. 250, 200, 150, 125 mmol/l : 5, 6. 100 mmol/l ;7, 8. 80 mmol/l ;9. 60 mmol/l ;10-12.

250, 200, 150 and 125 mmol/l imidazole elution; 5, 6. 100 mmol/l imidazole elution; 7, 8. 80 mmol/l imidazole elution; 9. 60 mmol/l imidazole elution; 10-12. 40 mmol/l imidazole elution; 13.

4 Fig. 5 5 rlm-phb2 Elution and purification of rlm-phb2 by His system A. rlm-phb2 ;M.protein marker; , 200, 150, 125 mmol/l : 5, mmol/l ;7, mmol/l ;9. 60 mmol/l ; mmol/l ; mmol/l ; B. rlm-phb2 M.protein marker;1. rlm-phb2 ; 2. rlm-phb2 ;3. rlm-phb2 A. Elution with different concentrations of imidazole M.protein marker; , 200, 150 and 125 mmol/l imidazole elution; 5, mmol/l imidazole elution; 7, mmol/l imidazole elution; mmol/l imidazole elution; mmol/l imidazole elution; mmol/l imidazole elution; B. purification of rlm-phb2 M. protein marker; 1.rLm-PHB2 proteins before induced; 2. rlm-phb2 proteins after induced; 3. Purified rlm-phb2 protein 6 rlm-phb2 Western blotting Fig. 6 Identification of rlm-phb2 by Western blotting M. marker;1. rlm-phb2 ; 2. rlm-phb2 M.low molecular weight protein marker;1.non-induced expression of Rosetta blue/pet-32a-lm-phb2;2.induced expression of Rosetta blue/pet-32a-lm-phb2 3 讨论与结论 PHB, Jang [14] PHB Gamble [15] PHB 50% PHB [16], PHB PHB,,,, PHB, [17] PHB [12-13], ROS,, PHB [12-13],,, [18] Liu [18], PHB, PHB, A PHB,, [19-20], IL6 TNF-a mapk, PHB [21] PHB PHB, Sripathi [22] PHB, PHB,, PHB2, rlm-phb2, rlm-phb2 PHB2, rlm-phb2 PHB [13], PHB PHB2,, pet-32a rlm-phb2, His,, 40 / 2015 / 39 / 4

5 pet-32a,, rlm-phb2, : [1],,,. [J]., 2010, 22(5): [2] Nuell M J, Stewart D A, Walker L, et al. PHB, an evolutionarily conserved intracellular protein that block DNA synthesis in normal fibroblasts and Hela cells[j]. Mol Cell Biol, 1991, 11(3): [3] Upe E R, Liu X T, Kiehlbauch J L, et al. Prohibitin antiproliferative activity and lack of heterozygosity in immortalized cell lines[j]. Exp Cell Res, 1995, 218(2): [4] Roskams A J, Friedman V, Wood C M J, et al. Cell cycle activity and expression of prohibitin mrna[j]. J Cell Physiol, 1993, 157(2): [5] Wang S, Nath N, Fusaro G, et al. Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals[j]. Molecular and Cellular Biology, 1999, 19(11): [6] Joshi B, Ko D, Ordonez-Ercan D, et al. A putative coiled-coil domain of prohibitin is sufficient to repress E2F1-mediated transcription and induce apoptosis[j]. Biochem Biophys Res Commun, 2003, 312(2): [7] Fusaro G, Dasgup T P, Rastogi S, et al. PHB induces the transcriptional activity of 0p53 and is exported from the nucleus upon apoptotic signaling [J]. J Biol Chem, 2003, 278(48): [8],,,. Rg1 MG-63 Prohibitin [J] , 24(2): [9] Fusaro G, Wang S, Chellappan S. Differential regulation of Rb family proteins and prohibitin during camp tothecin-induced apoptosis[j]. Oncogene, 2002, 21(29): [10] Wang S, Zhang B H, Faller D V. Prohibitin requires Brg-1 and Brm for the repression of E2F and cell growth[j]. The EMBO Journal, 2002, 21(12): [11] Choi D, Lee S J, Hong S, et al. Prohibitin interacts with RNF2 and regulates E2F1 function via dual pathways [J]. Oncogene, 2008, 27(12): [12],,,. [J]., 2013, 10: [13],,. [J]., 2010, 30(21): [14] Jang J S, Cho H Y, Lee Y J, et al. The differential proteome profile of stomach cancer identification of the biomarker candidates[j]. Oncology Res, 2004; 14: [15] Gamble S C, Odontiadis M, Waxman J, et.al Androgens target prohibitin to regulate proliferation of prostate cancer cells[j]. Oncogene, 2004: 23(17): [16] Wu T F, Wu H, Wang Y W, et al. Prohibitin in the pathogenesis of transitional cell bladder cancer[j]. Andcaneer Res, 2007; 27(2): [17],,. Prohibitin [J]., 2005, 25(3): [18] Liu X H, Ren Z, Zhan R, et al. Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte[j]. Cell Stress and Chaperones, 2009, 14: [19] Muraguchi T, Kawawa A, Kubota S. Prohibitin protects against hypoxia-induced H9c2 cardiomyocyte cell death[j]. Biomed Res, 2010, 31: [20],. ⅡA [J]., 2011, 6: [21] Rajalingam K, Wunder C, Brinkmann V, et al. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration[j]. Nat Cell Biol, 2005, 7(8): [22] Sripathi S R, He W, Atkinson C L, et al. Mitochondrial-Nuclear communication by prohibitin shuttling under oxidative stress[j]. Biochemistry, 2011, 50: Marine Sciences / Vol. 39, No. 4 /

6 Prokaryotic expression and purification of recombined lamprey PHB2 protein LI Tie-song, WANG Ying, GAO Yang, LI Qing-wei (Life Science College of Liaoning Normal University, Dalian , China) Received: Mar., 10, 2014 Key words: marine biology; lamprey; PHB2; prokaryotic expression; protein purification Abstract: To study the in vivo biological activities of lamprey PHB2 protein and prepare the antibody of PHB2 protein, herein, we aimed to construct the soluble prokaryotic expression vector of Lm-PHB2, and obtain adequate amount of purified rlm-phb2 protein. The Lm-PHB2 gene was amplified from the previously constructed pmd18-t-lm-phb2 plasmid template. The PCR products were subjected to Hind III and EcoR I digestion and then linked to a soluble expression vector pet-32a. The identified recombinant plasmid pet-32a-lm-phb2 was transformed into Rosetta Blue, and IPTG induced expression of rlm-phb2 was confirmed by SDS-PAGE and Western blotting assay. The rlm-phb2 fusion protein was purified using His affinity chromatography purification system to get highly purified protein. The correct construction of recombinant plasmid pet-32a-lm-phb2 was confirmed by PCR, restriction enzyme digestion and gene sequencing identification. Expression products were verified to present in the supernatant of bacteria lysis, indicting the successful soluble expression of rlm-phb2. The SDS-PAGE and Western blotting results showed that the molecular weight of rlm-phb2 protein was about 47 ku, corresponding with the anticipant size. The pet-32a-lm-phb2 prokaryotic expression vector was constructed correctly, and the soluble rlm-phb2 protein was successfully expressedsucceeded. Ultimately, the rlm-phb2 protein with high purity was obtained after purification. ( 本文编辑 : 谭雪静 ) 42 / 2015 / 39 / 4

材料方法

材料方法生物技术通报张发洲杨慧兰为了构建单纯疱疹病毒型感染细胞多肽真核表达质粒应用技术从株的基因组中扩增基因并连接至真核表达载体对阳性克隆进行菌落酶切和测序鉴定后成功构建了重组质粒用转染试剂盒将重组质粒转染至细胞中并用及检测其表达情况结果显示基因在细胞中得到正确表达真核表达质粒的构建成功为进一步研究对宿主细胞的影响奠定了基础单纯疱疹病毒型感染细胞多肽