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中国生物工程杂志

China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (2): 26-32    DOI: 10.13523/j.cb.20170205
    
Soluble Fusion-expression and Antitumor Activity Analysis of Apoptin of Chicken Anemia Virus
JI Jun, ZHU Chen-chen, XU Xin, LIU Xiau, LENG Chao-liang, SHI Hong-fei, YAO Lun-guang, KAN Yun-chao
Center for Nanyang Veterinary Biological Engineering Technology/Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Nanyang 473061, China
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Abstract  

To obtain high-efficiency soluble expressed apoptin gene of chicken anemia virus, the apoptin gene sequence was optimized based on E. coli codon preference and inserted into pBCX plasmid vector harbored msyB chaperonin, and then transformed into the competent into E. coli BL21 (DE3) to induce expression, followed by identification of SDS-PAGE and purified by nickel column affinity chromatography. The activity of anti-tumor was evaluated using microscopic examination, CCK 8 experimental determination and DNA ladders tests. The results showed that the E. coli expression vector pBCX-CAV-Apoptin was successful constructed. A large amount of soluble expression product was obtained in E. coli under the normal cultuered condition at 37℃. The fusion protein weighted 42 kDa, and 1.5 mg of purified recombinant proteins could be obtained from 50 ml bacteria liquid. CCK 8 experiment results showed that the purified recombinant protein could induce the growth inhibition rate over 60% of lymphoma JEKO-1 and REC-1 cells post treated 36 h. Microscope and DNA ladders tests intimated that the recombinant protein could induced the apoptosis of JEKO-1 and REC-1 cells rather than HUVEC cells. Western blotting tests indicated that the recombinant protein activated the expression of caspase-3, but not the cleavage of caspase-8 in JEKO-1 cells. It showed that the fusion protein msyB-CAV-apoptin could achieve efficient soluble expression, and the expressed products had specific antitumor activity.



Key wordsChicken anemia virus      Fusion chaperonin      Apoptin      Efficient soluble expression      Activity assay     
Received: 08 August 2016      Published: 25 February 2017
ZTFLH:  Q789  
Cite this article:

JI Jun, ZHU Chen-chen, XU Xin, LIU Xiau, LENG Chao-liang, SHI Hong-fei, YAO Lun-guang, KAN Yun-chao. Soluble Fusion-expression and Antitumor Activity Analysis of Apoptin of Chicken Anemia Virus. China Biotechnology, 2017, 37(2): 26-32.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170205     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I2/26

[1] Fearnhead H O, Rodriguez J, Govek E E, et al. Oncogene-dependent apoptosis is mediated by caspase-9. Proc Natl Acad Sci USA, 1998, 95(23):13664-13669.
[2] Hanahan D, Weinberg R A. Hallmarks of cancer:the next generation. Cell, 2011, 144(5):646-674.
[3] Singh P K, Tiwari A K, Rajmani R S, et al. Apoptin as a potential viral gene oncotherapeutic agent. Appl Biochem Biotechnol, 2015, 176(1):196-212.
[4] Saxena S, Kumar R, Singh P, et al. Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies. Indian J Exp Biol, 2012, 50(5):325-331.
[5] Maddika S, Booy E P, Johar D, et al. Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci, 2005, 118(pt19):4485-4493.
[6] Kuusisto H V, Wagstaff K M, Alvisi G, et al. The C-terminus of apoptin represents a unique tumor cell-enhanced nuclear targeting module. Int J Cancer, 2008, 123(12):2965-2969.
[7] Lin B, Kolluri S K, Lin F, et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell, 2004, 116(4):527-540.
[8] Poon I K, Oro C, Dias M M, et al. Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells. Cancer Res, 2005, 65(16):7059-7064.
[9] Rollano Peñaloza O M, Lewandowska M, Stetefeld J, et al. Apoptins:selective anticancer agents. Trends Mol Med, 2014, 20(9):519-528.
[10] 张艳玲, 徐 霞, 江露含, 等. 凋亡素原核表达载体构建及活性测定. 中国组织工程研究, 2015, 19(18):2928-2932. Zhang Y L, Xu X, Jiang L H, et al. Construction of a prokaryotic expression vector for apoptin and activity determination. Chinese Journal of Tissue Engineering Research, 2015, 19(18):2928-2932.
[11] 许鑫, 仲雨微, 李其久, 等. TAT-Apoptin 蛋白制备及其诱导BGC-823细胞的凋亡作用. 南开大学学报(自然科学版), 2015, 48(5):33-37. Xu X, Zhong Y W, Li Q J, et al. TAT-Apoptin protein preparation and induction of apoptosis of BGC-823 cell. Acta Scientiarum Naturalium Universitatis Nankaiensis, 2015, 48(5):33-37.
[12] Xie Q M, Ji J, Du L Q, et a. Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine. Poult Sci, 2009, 88(8):1608-1615.
[13] Jin J L, Gong J, Yin T J, et al. PTD4-apoptin protein and dacarbazine show a synergistic antitumor effect on B16-F1 melanoma in vitro and in vivo. Eur J Pharmacol, 2011, 654(1):17-25.
[14] Pennant W A, An S, Gwak S J, et al. Local non-viral gene delivery of apoptin delays the onset of paresis in an experimental model of intramedullary spinal cord tumor. Spinal Cord, 2014, 52(1):3-8.
[15] Yuan L, Zhao H, Zhang L, et al. The efficacy of combination therapy using adeno-associated virus-mediated co-expression of apoptin and interleukin-24 on hepatocellular carcinoma. Tumour Biol, 2013, 34(5):3027-3034.
[16] Ma J L, Han S X, Zhao J, et al. Systemic delivery of lentivirus-mediated secretable TAT-apoptin eradicates hepatocellular carcinoma xenografts in nude mice. Int J Oncol, 2012, 41(3):1013-1020.
[17] Wu Y, Zhang X, Wang X, et al. Apoptin enhances the oncolytic properties of Newcastle disease virus. Intervirology, 2012, 55(4):276-286.
[18] Sun J, Yan Y, Wang X T, et al. PTD4-apoptin protein therapy inhibits tumor growth in vivo. Int J Cancer, 2009, 124(1):2973-2981.
[19] 郭争荣, 彭焕彦, 亢继文,等. 细胞穿透肽:一种新型非病毒载体. 中国生物工程杂志, 2016, 36(6):100-106. Guo Z R, Peng H Y, Kang J W, et al. Cell penetrating peptides:research progress of a novel non-viral vectors. China Biotechnology, 2016, 36(6):100-106.
[20] 刘雪梅, 崔剑, 侯伟健, 等. TAT-Apoptin融合蛋白分泌表达载体的构建及其活性检测. 中国医科大学学报, 2013, 42(1):45-48. Liu X M, Cui J, Hou W J, et al. Construction of prokaryotic secretory expression vector of TAT-apoptin and expression in E. coli. Journal of China Medical University, 2013, 42(1):45-48.

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