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| Prokaryotic Expression and Purification of VEGF from Sika Deer (Cervus nippon) |
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Abstract Velvet is a natural cell growth factor library. The top tissue of the antler as the most active growth point of the deer body to store a large number of growth factors, they are the biochemical basis of the clinical role of deer antler. A variety of growth factors have important roles in promoting the proliferation of antler. The rapid growth of velvet and the generation of blood vessels in cartilage tissue are inseparable and vascular endothelial growth factor (VEGF) can promote blood cell formation and promote the formation of blood vessels. Therefore, the study of sika deer (Cervus nippon) VEGF will provide a reference for the mechanism of rapid growth of velvet. To obtain the biologically active protein, the pET-30a prokaryotic expression vector and pMD-18T-VEGF were digested with HindⅢ and BamHⅠ. The digested product was recovered separately. VEGF mature peptide gene from Cervus nippon was inserted to expression vector pET-30a by T4 DNA ligase to obtain recombinant plasmid. The recombinant plasmids were identified by PCR and identified by double digestion. The correct recombinant plasmid was made into glycerol. Proteins were expressed (0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 20 ℃) in the host Rosetta cells in this study. The results were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Target protein was purified by Ni-Agarose affinity column and renatured to obtain the biologically active protein. Target protein on the proliferation of mouse embryonic fibroblast cell line (NIH3T3) cell was analysed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). A specific band of about 519 bp was obtained by double digestion of pET-30a-VEGF with 2 restriction endonucleases BamHⅠ and HindⅢ, which were consistent with the expected results. The PCR results of pET-30a-VEGF were used as templates to show a band corresponding to the size of the target band. The results showed that the VEGF gene of sika deer was successfully inserted into pET-30a vector. Restriction enzyme mapping showed that pET-30a-VEGF expression vector was constructed successfully. The recombinant plasmid pET-30a-VEGF was transformed into Escherichia coli Rosetta competent cells and induced by IPTG. SDS-PAGE results showed that there was a specific band at about 23.4 kD as shown, which was consistent with the expected results. Optimization of the optimal IPTG concentration showed that the protein was highest expressed when the IPTG concentration was 0.6 mmol/L at 37 ℃. The best IPTG induction temperature was optimized, and when the temperature was 20 ℃, the protein was the highest expressed. The induction time was optimized. The results showed that the expression level of recombinant protein was the highest when the induction time was 16 h. Therefore, when the IPTG concentration was 0.6 mmol/L, the induction temperature was 20 ℃ and the induction time was 16 h, the recombinant protein was the highest. The purified VEGF mature peptide appeared at a molecular mass of about 23.4 kD, which was consistent with the molecular weight of VEGF mature peptide. The results of MTT showed that proliferation of NIH3T3 was promoted by different concentration of recombinant protein, and the proliferation rate of the cells increased with the increase of the concentration. Among them, the most significant impact on NIH3T3 cell proliferation was at 48 h, and the final concentration was 200 ng/mL. The results of the study will further explore the function of VEGF in the rapid value-added process of velvet and regulation mechanism to provide experimental basis.
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Received: 02 March 2017
Published: 16 June 2017
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| [1]才蕾,矫丽媛.甲胎蛋白的原核表达及复性优化[J].生物技术通报,2015,31(1):193-197.(Cai L, Jiao L Y. Prokaryotic expression of alpha fetoprotein and optimization of renaturation[J]. Biotechnology Bulletin,2015,31(1):193-197.)[2]郝林琳,张永亮.鹿茸多肽研究及IGF-1 5’端调控序列分析[D].长春:吉林大学,2006.(Hao L L, Zhang Y L. Study on deer velvet antler polypeptides and analysis of IGF-1 5’ end regulatory sequence[D].Changchun:Jilin University,2006.)[3]胡薇,郝军元,艾永兴,等.人血管内皮生长因子和端粒酶催化亚基调控序列的构建及转录活性的研究[J].吉林农业大学学报,2006,28(2):164-167.(Hu W, Hao J Y, Ai Y X , et al. Construction of multiplicity transcriptional regulatory elements of vascular endotrelial growth factor enhancer and telomerase catatytic submit core promoter and research of transcription activity[J]. Journal of Jilin Agricultural University,2006,28(2):164-167.)[4]胡薇,孟星宇,田玉华,等.梅花鹿IGF1全长cDNA克隆及在鹿茸组织的表达[J].东北林业大学学报,2011,39(11):71-75.(Hu W, Meng X Y, Tian Y H, et al. Cloning of a full-length cDNA encoding Insulin-Like growth factor 1 and its expression in antler tissue[J].Journal of Northeast Forestry University,2011,39(11):71-75.)[5]胡薇,牛永梅,司博,等.原核表达梅花鹿IGF-1成熟多肽对鸡CEF和CEK细胞增殖的影响[J].吉林农业科学,2015,40(4):68-71,112.(Hu W, Niu Y M, Si B, et al. Effect of prokaryotic expression of IGF-1 mature peptide from cervus nippon on proliferation of chicken CEF and CEK cells[J]. Journal of Jilin Agricultural Science,2015,40(4):68-71,112.)[6]李春义.鹿茸完全再生机制研究进展[J].农业生物技术学报,2017,25(1):1-10.(Li C Y. Progress on the mechanism underlying full regeneration of mammalian organ deer antlers[J].Journal of Agricultural Biotechnology,2017,25(1):1-10.)[7]李骏,刘旭东.浅谈VEGF的相关研究进展[J].中国继续医学教育,2016,8(9):201-203.(Li J, Liu X D.The research progress of VEGF[J].China Continuing Medical Education,2016,8(9):201-203.)[8]刘侃,汪炬,谢秋玲,等.重组类胰岛素样生长因子-1的纯化与复性[J].中国生物工程杂志,2006,26(2):29-33.(Li K, Wang J, Xie Q L, et al. Purification and refolding of recombinant human insulin - like growth factor 1[J]. China Biotechnology,2006,26(2):29-33.)[9]李璐.microRNA-93-5p与microRNA-20b-5p对VEGF基因转录调控作用与鹿茸细胞增殖关系的研究[D].吉林:吉林农业大学,2015.(Li L.Study of microRNA-93-5p and microRNA-20b-5p on the relationship with VEGF transcriptional regulation and deer antler cell proliferation[D].Jilin:Jilin Agricultural University,2015.)[10]李璐,李婷,李沐,等.miRNA-93-5p对VEGF的转录调控及其与梅花鹿茸细胞增殖的关系[J].东北林业大学学报,2014,42(11):146-149.(Li L, Li T, Li M, et al. Transcription regulation effect of miRNA-93-5p on VEGF gene and its relationship with antler cell proliferation[J]. Journal of Northeast Forestry University,2014,42(11):146-149.)[11]李玉灵,赵华,任秀宝.VEGF及其受体与免疫抑制细胞关系的研究进展[J].山东医药,2016,56(17):105-107.(Li Y L, Zhao H, Ren X B. Research progress of immunosuppressive cells relationship between VEGF and its receptors[J]. Shandong Medical Journal ,2016,56(17):105-107.)[12]孟星宇,李婷,李沐,等.梅花鹿IGF-1的原核表达与纯化[J].西北农林科技大学学报(自然科学版),2012,40(7):7-13.(Meng X Y, Li T, Li Mu, et al. Expression and purification of IGF-1 from cervus nippon[J]. Journal of Northwest A&F University(Nat, Sci, Ed.),2012,40(7):7-13.)[13]王丛笑,宋军营,张振强.VEGF在阿尔茨海默病重相关作用机制的研究[J].中医学报,2015,30(200):109-113.(Wang C X, Song J Y, Zhang Z Q, et al. VEGF related mechanisms in alzheimer’s disease research[J].China Journal of Chinese Medicine,2015,30(200):109-113.)[14]吴炎.梅花鹿VEGF基因的克隆、原核表达及对细胞增殖的影响[D].吉林:吉林农业大学,2015.(Wu Y. Study on cloning expression and biological activity of VEGF gene from skia deer[D].Jilin:Jilin Agricultural University,2015.)[15]夏海峰,张显.镍离子亲和层析介质的制备及其用于组氨酸标记蛋白质的纯化[J].江南大学学报,2010,9(6):685-689.(Xia H F, Zhang X. Preparation of Ni affinity chromatographic adsorbent and its application on purification of His-tagged protein[J]. Journal of Jiangnan University(Natural Science Edition),2010,9(6):685-689.)[16]闫语多,韩香玉,吴雷,等.鹿茸组织microRNA的研究进展[J].中国畜牧杂志,2016,52(21):90-94.(Yan Y D, Han X Y, Wu Lei, et al. Advances in microRNA in antler velvet tissues[J].Chinese Journal of Animal Science, 2016,52(21):90-94.)[17]赵静,闫伟,雷平.VEGF和PEDF对血管渗透性影响的研究进展[J].中国社区医师,2016,32(31):7-9.(Zhao J, Yan W, Lei P. Research progress on the effect of VEGF and PEDF on vascular permeability[J].Chinese Community Doctors,2016,32(31):7-9.)[18]Iacovelli R,Sternberg CN,Porta C,et al.Inhibition of the VEGF / VEGFR pathway improves survival in advanced kidney cancer: a systematic review and meta-anly-sis [J].Curr Drug Targets,2015,16(2):164-170.[19]Kierdorfa U,LI C Y,Price J S,et al. Improbable appendages: deer antler renewal as a unique case of mammalian regeneration[J]. Semin Cell Dev Biol,2009,20(5) : 535-542.[20]Li C,Suttie J.Morphogenetic aspects of deer antler development[J].Frontiers in Bioscience(Elite Edition),2012,4(1):1836-1842.[21]Lau NC,Lim LP,Weinstein EG,et al.An abundant class of tiny RNAs with probable regulatory roles in Caenor habditiselegans[J].Science,2001,294(5543):858-863.[22]Park HJ,Lee DH,Park SG,et al.Proteome analysis of red antlers[J].Proteomics,2004,4(11):3642-3653. |
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