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EB病毒融合蛋白Zta-P54在大肠杆菌中的表达、纯化及鉴定

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Keywords: EB病毒,Zta-P54融合蛋白,原核表达,蛋白纯化

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Abstract:

为获得能用于检测试剂盒的高纯度具有活性的EB病毒融合蛋白,以pGEX5T-BZLF1-BMRF1质粒为模板进行PCR扩增得到BZLF1-BMRF1融合基因,将其插入pET32a中,构建表达质粒pET32a/BZLF1-BMRF1。将该质粒转入大肠杆菌培养,经IPTG诱导获得Zta-P54融合蛋白。用DEAE-SepharoseCL-6B和Ni-NTA亲和层析纯化,并通过SDS-PAGE和Westernblot对Zta-P54融合蛋白进行鉴定。双酶切鉴定和测序结果显示成功构建pET32a/BZLF1-BMRF1质粒,SDS-PAGE显示该蛋白相对分子量约为60kD,与预期结果一致。纯化后获得纯度为96.5%的Zta-P54融合蛋白。Westernblot检测该蛋白有良好的生物活性和反应特异性。因此,成功构建pET32a/BZLF1-BMRF1质粒,在大肠杆菌中可溶性表达。最终获得纯度高、生物活性好的融合蛋白。

References

[1]  Fachiroh J, Stevens SJ, Haryana SM, et al. Combination of Epstein-virus scaffold(BDRF1/VCA-p40)and small capsid protein(BFRF3/VCA-p18)into a single molecule for imprived serodiagnosis of acute and malignant EBV-driven disease[J]. J Virol Methods, 2010, 169(1), 79-86.
[2]  张毅, 周淑艳, 孙业红, 等.EB病毒融合蛋白 ztaN-p23 在大肠杆菌中的表达、纯化及活性鉴定[J].细胞与分子免疫学杂志, 2012, 28(2):120-123.
[3]  伊强.以原核表达的EB病毒融合蛋白p54-ztaN为抗原建立ELISA方法及其在鼻咽癌诊断中的应用[D] .福建:福建医科大学, 2008.
[4]  Feederle R, Kost M, Baumann M, et al. The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators[J]. EMBO J, 2000, 19(12):3080-3089.
[5]  顾耀亮, 张昌卿, 吴子柏, 等.抗重组EB病毒双重抗体鼻咽癌血清学研究[J].癌症, 2003, 22(9):903-906.
[6]  Islam MS, Anoop P, Gordon-Smith EC, et al. Epstein-Barr virus infections after allogeneic stem cell transplantation A comparison between nonmalignant and malignant hematological disorder[J]. Hematology, 2010, 15(5):344-350.
[7]  Chan KH, Gu YL, Ng F, et al. EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma[J]. Cancer, 2003, 105(5):706-709.
[8]  Paramita DK, Fachiroh J, Artama WT, et al. Native early antigen of Epstein-Barr virus a promissing antigen for diagnosis of nasopharyngeal carcinoma[J]. J Med Virol, 2007, 79(11):1710-1721.
[9]  Dardari R, Hinderer W, Lang D, et al. Antibody responses to recombinant Epstein-Barr virus antigens in nasopharyngeal carcinoma patients:Complementary test of ZEBRA protein and early antigens p54 and p138[J]. J Clin Micro, 2001, 39(9):3164-3169.
[10]  Chan KH, Gu YL, Ng F, et al. EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma[J]. Int J Cancer, 2003, 105(4):706-709.
[11]  Chan JKC, Pilch BZ, Bray F, et al. Nasopharyngeal carcinoma[M] //Barnes L, Eveson JW, Reichart P, Sidransky D. World Health Orga-nization Classification of Tumours. Pathology&Genetics. Head and Neck Tumours. Lyon France:IARC-Press, 2005:85-97.
[12]  Allen MD, Young LS, Dawson CW. The Epstein-virus encoded LMP2A and LMP2B proteins promote epithelial cell spreading and motility[J]. Virol, 2005, 79(6):1789-1802.
[13]  伊强, 王文华, 陈丹, 等.以合成肽为抗原建立ELISA试验检测鼻咽癌患者EB病毒抗体的研究[J].中华肿瘤防治杂志, 2008, 15(8):610-611.
[14]  Hoebe EK, hutajulu SH, VanBeek J, et al. Purified hexameric Epstein-virus encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients[J]. Clin Vaccine Immunol, 2011, 18(2):298-304.

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