黄夏冰,康巧珍,傅国,汲振余,刘鑫

• 1:郑州大学生命科学学院分子免疫学实验室
• 2:河南省医药科学研究院

摘要(Abstract)：

利用基因工程的方法,以实验室保存的p MAL-C2X-NAP质粒为模板,PCR扩增NAP基因,构建重组质粒p GEX-NAP.重组质粒通过酶切和测序鉴定后转化大肠杆菌表达菌株BL21(DE3),再经IPTG低温诱导获得可溶性GST-NAP融合蛋白,最后利用谷胱甘肽琼脂糖凝胶树脂进行纯化,Prescission蛋白酶进行柱上切割去除GST标签.结果表明,p GEX-NAP重组质粒构建正确,在大肠杆菌中经IPTG低温诱导表达,可获得大量可溶性GST-NAP融合蛋白.Prescission蛋白酶柱上切割去除GST标签后,经Western Blot验证NAP蛋白能被兔抗NAP多克隆抗体特异识别.

关键词(KeyWords)： GST-NAP;重组质粒;蛋白表达;GST标签

Abstract:

Keywords:

基金项目(Foundation): 重大新药创制科技重大专项基金项目,编号2012ZX09103301-022;; 国家自然科学基金资助项目,编号U1204817,81373119

作者(Author): 黄夏冰,康巧珍,傅国,汲振余,刘鑫

参考文献(References)：

• [1]Satin B,Del P G,Della Bianca V,et al.The neutrophil-activating protein(HP-NAP)of Helicobacter pylori is a protective antigen and a majorvirulence factor[J].The Journal of Experimental Medicine,2000,191(9):1467-1476.
• [2]Amedei A,Cappon A,Codolo G,et al.The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses[J].The Journal of Clinical Investigation,2006,116(4):1092-1101.
• [3]Del P G,Chiumiento L,Amedei A,et al.Immunosuppression of Th2 responses in Trichinella spiralis infection by Helicobacter pylori neutrophil-activating protein[J].The Journal of Allergy and Clinical Immunology,2008,122(5):908-913.
• [4]Amedei A,Codolo G,Del P G,et al.The effect of Helicobacter pylori on asthma and allergy[J].Journal of Asthma and Allergy,2010,3:139-147.
• [5]de Bernard M,D'Elios M M.The immune modulating activity of the Helicobacter pylori HP-NAP:Friend or foe?[J].Toxicon:Official Journal of the International Society on Toxinology,2010,56(7):1186-1192.
• [6]Codolo G,Mazzi P,Amedei A,et al.The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma[J].Cellular Microbiology,2008,10(11):2355-2363.
• [7]Codolo G,Fassan M,Munari F,et al.HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response[J].Cancer Immunology,Immunotherapy:CII,2012,61(1):31-40.
• [8]Ramachandran M,Yu Di,Wanders A,et al.An infection-enhanced oncolytic adenovirus secreting H.pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors[J].Molecular Therapy:the Journal of the American Society of Gene Therapy,2013,21(11):2008-2018.
• [9]Fu Huawen.Helicobacter pylori neutrophil-activating protein:From molecular pathogenesis to clinical applications[J].World Journal of Gastroenterology,2014,20(18):5294-5301.
• [10]张帅清.如何提高幽门螺杆菌培养阳性率[J].中国卫生检验杂志,2007,17(4):759-760.
• [11]李莉,贾新成,秦广雍,等.酵母海藻糖合成酶基因植物表达载体的构建[J].郑州大学学报:理学版,2003,35(1):49-51.
• [12]吴玉荣,吴海涛,曹萌,等.重组PET30a/hs BLy S质粒在BL21(DE3)菌株中的稳定性观察[J].信阳师范学院学报:自然科学版,2005,18(2):165-168.
• [13]Smith D B,Johnson K S.Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione Stransferase[J].Gene,1988,67(1):31-40.
• [14]段沁江.谷胱甘肽S-转移酶与肝脏病变研究的新进展[J].环境卫生学杂志,1995,22(1):8-11.
• [15]Wang Changsen,Castro A F,Wilkes D M,et al.Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product:comparison of fusions to glutathione S-transferase,thioredoxin and maltosebinding protein[J].Biochemical Journal,1999,338(1):77-81.
• [16]赵战勤,薛云,赵刚,等.重组百日咳杆菌黏附素C端蛋白的免疫原性[J].河南科技大学学报:自然科学版,2010,31(5):76-79.
• [17]Walker P A,Leong L E,Ng P W P,et al.Efficient and rapid affinity purification of proteins using recombinant fusion proteases[J].Biotechnology,1994,12(6):601-605.