重组蓖麻毒素A链的表达及半乳糖修饰
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摘要
目的表达重组蓖麻毒素A链(ricin toxin A,RTA),并进行半乳糖糖基化修饰。方法将重组质粒pET-28a-RTA转化E. coli BL21(DE3),经IPTG诱导表达重组蛋白RTA,通过镍离子亲和层析柱纯化包涵体,并复性。采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC]/N,N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)法对重组RTA进行半乳糖糖基化修饰,并对偶联反应温度(20、25、30和37℃)和半乳糖与RTA的投料比(100∶1、500∶1、1 000∶1、1 500∶1和2 000∶1)进行优化。结果 RTA蛋白相对分子质量约34 000,主要以包涵体形式表达,纯化复性后纯度大于90%。最适偶联反应温度为25℃,半乳糖与RTA最适投料比为1 000∶1。结论成功表达了RTA蛋白,并通过EDC/NHS法实现了半乳糖与RTA的偶联。
Objective To express recombinant ricin toxin A chain(RTA) and modify by glycosylation with galactose.Methods Recombinant plasmid pET-28 a-RTA was transformed to E. coli BL21(DE3)and induced by IPTG. The expressed RTA was purified by nickel ion affinity chromatography and refolded,then modified by galactose glycosylation using 1-(3-dimethylaminoprophyl)-3-ethylcarbodiimide hydrochloride(EDC)/N-hydroxysuccinimide(NHS),and the temperature for coupling reaction(20,25,30 and 37 ℃) and the ratio of galactose to RTA(100 ∶ 1,500 ∶ 1,1 000 ∶ 1,1 500 ∶ 1 and 2 000 ∶ 1)were optimized. Results The expressed RTA with a relative molecular mass of about 34 000 was mainly in a form of inclusion body,of which the purity was more than 90%. The optimal temperature for coupling reaction was25 ℃,while the optimal ratio of galactose to RTA was 1 000 ∶ 1. Conclusion RTA was expressed successfully and coupled to galactose by EDC/NHS method.
Objective To express recombinant ricin toxin A chain(RTA) and modify by glycosylation with galactose.Methods Recombinant plasmid pET-28 a-RTA was transformed to E. coli BL21(DE3)and induced by IPTG. The expressed RTA was purified by nickel ion affinity chromatography and refolded,then modified by galactose glycosylation using 1-(3-dimethylaminoprophyl)-3-ethylcarbodiimide hydrochloride(EDC)/N-hydroxysuccinimide(NHS),and the temperature for coupling reaction(20,25,30 and 37 ℃) and the ratio of galactose to RTA(100 ∶ 1,500 ∶ 1,1 000 ∶ 1,1 500 ∶ 1 and 2 000 ∶ 1)were optimized. Results The expressed RTA with a relative molecular mass of about 34 000 was mainly in a form of inclusion body,of which the purity was more than 90%. The optimal temperature for coupling reaction was25 ℃,while the optimal ratio of galactose to RTA was 1 000 ∶ 1. Conclusion RTA was expressed successfully and coupled to galactose by EDC/NHS method.
引文
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[3]JETZT A E,LI X P,TUMER N E,et al.Toxicity of ricin Achain is reduced in mammalian cells by inhibiting its interaction with the ribosome[J].Toxicol Appl Pharm,2016,60(12):7153-7158.
[4]MOSHIRI M,HAMID F,ETEMAD L.Ricin toxicity:clinical and molecular aspects[J].Rep Biochem Mol Biol,2016,4(2):60-65.
[5]WORBS S,K HLER K,PAULY D,et al.Ricinus communis intoxications in human and veterinary medicine a summary of real cases[J].Toxins,2011,3(10):1332-1372.
[6]COWARDIN C A,JACKMAN B M,NOOR Z,et al.Glucosylation drives the innate inflammatory response to Clostridium difficile toxin A[J].Infect Immun,2016,84(8):2317-2323.
[7]YAN Q,LI X P,TUMER N E.N-glycosylation does not affect the catalytic activity of ricin A chain but stimulates cytotoxicity by promoting its transport out of the endoplasmic reticulum[J].Traffic,2012,13(11):1508-1521.
[8]LIU Z,LI J,LI J,et al.Mannan-modified Ad5-PTEN treatment combined with docetaxel improves the therapeutic effect in H22tumor-bearing mice[J].Int J Nanomed,2012,9(7):5039-5049.
[9]BAI RB,MA YL,ZHANG P,et al.Content determination of saccharide in polysaccharides containing galacturonic acid by phenolsulfuric acid method combined with calibration factor method[J].Chin Prac Med,2017,28(21):2974-2978.(in Chinese)白瑞斌,马玉玲,张培,等.苯酚-硫酸法结合校正因子法测定含半乳糖醛酸的多糖中的糖含量[J].中国药房,2017,28(21):2974-2978.
[10]RATHORE U,SAHA P,KESAVARDHANA S,et al.Glycosylation of the core of the HIV-1 envelope subunit protein gp120is not required for native trimer formation or viral infectivity[J].J Biol Chem,2017,292(24):10197-10219.
[11]XU C X,LU T C,WANG X L,et al.Biological activity of mannoligosaccharide-modified Rsαprotein of Brucella[J].Chin J Biologicals,2017,30(1):11-14.(in Chinese)许春晓,卢天成,王晓龙,等.甘露寡糖修饰布氏菌Rsα蛋白的生物活性[J].中国生物制品学杂志,2017,30(1):11-14.
[12]ZHU L.The glycosylation modification analysis of CTLA4-Ig fusion protein and the study of its influence on the stability and function[D].Shanghai:The Second Military Medical University,2016.(in Chinese)朱磊.重组CTLA4-Ig融合蛋白的糖基化修饰分析及其对稳定性和功能影响的研究[D].上海:第二军医大学,2016.
[13]XU X L,LI X S,WANG Q,et al.Uptake of norcantharidingalactose-modified chitosan nanoparticles by human liver tumor cells SMMC-7721 and HepG2 and effects of anti-tumor activity in S180 tumor-bearing mice[J].Chin J Gerontol,2017,37(15):3661-3664.(in Chinese)徐晓莉,李晓森,王钦,等.人肝肿瘤细胞SMMC-7721、HepG2对去甲斑蝥素-半乳糖修饰壳聚糖纳米粒的摄入及S180荷瘤小鼠在体抗肿瘤活性的影响[J].中国老年学杂志,2017,37(15):3661-3664.
[14]ZHANG N,BU G H,ZHU T W,et al.Effect of glycosylation on the antigenicity and structural properties of soybean protein isolate-lactose conjugate[J].Modern Food Sci Technol,2015,31(8):117-121.(in Chinese)张楠,布冠好,朱婷伟,等.糖基化反应对大豆蛋白-乳糖复合物抗原性及结构的影响[J].现代食品科技,2015,31(8):117-121.
[15]YIN Y,ZHANG X,QIAO Y,et al.Glycosylation at 11Asn on hemagglutinin of H5N1 influenza virus contributes to its biological characteristics[J].J Vet Res,2017,48(1):81-90.