利用大肠杆菌高效制备人端粒酶逆转录酶抗原肽
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摘要
目的利用大肠杆菌基因工程系统建立人端粒酶逆转录酶h TERT抗原肽的高效制备方法.方法利用抗原表位预测软件SYFPEITHI、BIMAS与Epi Jen确定截短表达策略,通过Prot Scale软件亲水性分析改善表达产物的可溶性.目的基因克隆于表达载体p ET21b,高浓度尿素裂解法溶解包涵体蛋白,稀释复性法复性.结果 3个覆盖全长h TERT的截短片段仅Ser480-Leu665能高效表达,但所形成的包涵体蛋白难溶于8 mol/L尿素.进一步优化所获的Tyr562-Ile665包涵体蛋白则可溶,且经纯化后可有效复性,产物纯度达到95%以上.结论利用大肠杆菌系统成功建立了h TERT抗原肽高效制备方法,为后续肿瘤疫苗的开发应用奠定了基础.
Objective To establish an efficient preparation method of human telomerase reverse transcriptase antigen peptide by Escherichia coli genetic engineering system. Methods Epitope prediction softwares SYFPEITHI, BIMAS and Epi Jen were used to determine the truncated expression strategy, and the hydropathicity analysis of Prot Scale software was used to improve solubility of the expression product. Target DNA sequence was cloned into p ET-21 b,an expression vector,and then the inclusion bodies were dissolved in high concentration urea and refolded by dilution. Results Only Ser480-Leu665 of 3 truncated fragments covering full-length h TERT was efficient expressed. However,the inclusion bodies were difficult to dissolve in 8 mol/L urea. Further truncated h TERT(Tyr562-Ile665) was also expressed as inclusion bodies,which was dissolved in 8 mol/L urea as expected. The purified inclusion protein of h TERT(Tyr562-Ile665),which purity was above 95%,was refolded in 20 mmol/L Tris-HCl(pH8.2). Conclusion An efficient preparation method of human telomerase reverse transcriptase antigen peptide with recombinant Escherichia coli has been established,which lays a foundation for development and application of tumor vaccine.
Objective To establish an efficient preparation method of human telomerase reverse transcriptase antigen peptide by Escherichia coli genetic engineering system. Methods Epitope prediction softwares SYFPEITHI, BIMAS and Epi Jen were used to determine the truncated expression strategy, and the hydropathicity analysis of Prot Scale software was used to improve solubility of the expression product. Target DNA sequence was cloned into p ET-21 b,an expression vector,and then the inclusion bodies were dissolved in high concentration urea and refolded by dilution. Results Only Ser480-Leu665 of 3 truncated fragments covering full-length h TERT was efficient expressed. However,the inclusion bodies were difficult to dissolve in 8 mol/L urea. Further truncated h TERT(Tyr562-Ile665) was also expressed as inclusion bodies,which was dissolved in 8 mol/L urea as expected. The purified inclusion protein of h TERT(Tyr562-Ile665),which purity was above 95%,was refolded in 20 mmol/L Tris-HCl(pH8.2). Conclusion An efficient preparation method of human telomerase reverse transcriptase antigen peptide with recombinant Escherichia coli has been established,which lays a foundation for development and application of tumor vaccine.
引文
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[2]营孙阳,熊加秀,麦洪旭,等.端粒酶调控研究进展[J].遗传,2016,38(4):289-299.
[3]赵相轩,卢再鸣.端粒酶活性抑制在癌症治疗中的作用研究进展[J].现代肿瘤医学,2017,25(19):3178-3181.
[4]JANKNECHT R.On the road to immortality:h TERT upregulation in cancer cells[J].Febs Letters,2004,564(9):1-2
[5]RESCIGNO M,AVOGADRI F,CURIGLIANO G.Challenges and prospects of immunotherapy as cancer treatment[J].Biochim Biophys Acta,2007,1776(1):108-123.
[6]BRUSIC V,BAJIC V B,PETROVSKY N.Computational methods for prediction of T-cell epitopes--a framework for modelling,testing,and applications[J].Methods,2004,34(4):436-443.
[7]朱颐申,张霓.多肽疫苗研究进展[J].中国疫苗和免疫,2013,19(1):69-74.
[8]万岩岩,钟静静,高宁宁,等.肿瘤多肽疫苗的研究进展[J].动物医学进展,2015,36(2):101-105.
[9]熊佳,孙明立,魏敏杰.肿瘤相关抗原CTL表位DC疫苗优化策略研究进展[J].现代肿瘤医学,2017,25(16):2677-2681.
[10]慎华平.h TERT多表位肽致敏m DC诱导CTL对HLA-A24阳性肿瘤细胞杀伤效应的研究[D].重庆医科大学,2011.
[11]廖忠莉,汤旭东,王国珍,等.人端粒酶逆转录酶表位多抗原肽疫苗的体外抗肿瘤免疫效应研究[J].第三军医大学学报,2012,34(11):1035-1039.
[12]MIZUKOSHI E,NAKAGAWA H,KITAHARA M,et al.Immunological features of T cells induced by human telomerase reverse transcriptase-derived peptides in patients with hepatocellular carcinoma[J].Cancer Letters,2015,364(2):98-105.
[13]吕韵哲,孙建龙,陈孟谦,等.人端粒酶逆转录酶在大肠杆菌中表达的初步研究[J].复旦学报(自然科学版),2002,41(6):621-625.
[14]刘红梅.h TERT重组质粒的构建及其表达[D].郑州:郑州大学,2004.
[15]童茵,金洁,娄引军.h TERT1658-2070在大肠杆菌中的克隆及表达[J].肿瘤,2002,22(4):276-278.
[16]尚世彤.人类端粒酶逆转录酶基因的原核表达及其表达产物抗体的制备[D].天津:天津医科大学,2002.
[17]袁竞妍,王宇,刘博轩,等.人端粒酶逆转录酶抗原HLAA0201限制性CTL表位预测及鉴定[J].中华肺部疾病杂志(电子版),2017,10(3):257-262.
[18]de GROOT A S,SBAI H,AUBIN C S,et al.Immunoinformatics:mining genomes for vaccine components.[J].Immunol Cell Biol,2002,80(3):255-259.
[19]耿建,黄桂春,王锐,等.人端粒酶逆转录酶HLA-A*0201限制性CTL表位的预测和鉴定[J].医学研究生学报,2011,24(10):13-18.