新城疫病毒血凝素-神经氨酸酶蛋白的可溶性超表达与免疫活性检测

doi:10.16098/j.issn.0529-1356.2016.06.023

摘要/Abstract

摘要：

目的检测新城疫病毒（NDV）血凝素-神经氨酸酶(HN)蛋白在大肠杆菌中的可溶性表达量，及其免疫活性。方法采用融合表达的方法将HN蛋白基因分别与Grifin、谷胱甘肽S移酶（GST）、麦芽糖结合蛋白（MBP）、 Nus A、小泛素样相关修饰物（SUMO）、硫氧还蛋白(thioredoxin)、蛋白G（protein G)、γ-晶状体蛋白（γ-crystallin）、 Ars C、 Ppi B 10种融合标签基因采用柔性接头进行连接，构建10个重组表达质粒，经酶切及测序鉴定正确后，将这10个重组质粒分别转入大肠杆菌BL21中，用不同条件进行诱导表达，经SDS-PAGE检测并选出最佳诱导蛋白表达的条件，筛选出能够显著促进HN蛋白可溶性表达的标签，并用Western blotting对所表达蛋白的含量进行定性分析，同时纯化的重组蛋白用Western blotting和间接ELISA验证其免疫原性。结果成功构建了10个不同融合标签的HN融合蛋白表达载体，筛选出融合标签麦芽糖结合蛋白（MBP）能够显著促进HN蛋白可溶性表达，且Western blotting显示所表达重组蛋白的表达量是最多的。经Western blotting和间接ELISA验证重组HN蛋白具有良好的免疫原性。结论融合标签MBP可以显著提高HN蛋白在大肠杆菌中的可溶性表达量。

关键词: 新城疫病毒, 血凝素-神经氨酸酶, 融合标签, 柔性接头, 免疫印迹法, 间接ELISA, 鸡

Abstract:

Objective To improve the soluble overexpression of Newcastle disease virus(NDV) hemagylutinin-neuraminidase(HN) protein in E. Coli and determine its immunological. Methods Ten plasmids that expressed NDV HN protein with-N-terminal Grifin, gultathione-S-transferase(GST), maltose biniding protein(MBP), NusA, sanall ubiquitin-related modifier(SUMO), thioredoxin, protein G, γ-crystallin, Ars C or Ppi B tags were constructed to test the effects of the various tags on the expression level and solubility of NDV HN protein in E. coli. The sequences of the ten plasmids were confirmed and the ten plasmids were transformed into E. coli BL21 strain. In order to select and screen the best expression conditions, different induced dose isopropy-β-D-thiogalactoside(IPTG) final concentration, culture temperature, Amp final concentration and culture media were used. The expression level and solubility of fusion protein were analyzed by SDS-PAGE and Western blotting. The fusion proteins were purified by Ni-NTA chromatography to develop indirect ELISA. The purified fusion protein was detected by Western blotting and indirect ELISA. Results Ten HN fusion protein expression plasmids were constructed successfully. Maltose binding protein(MBP) tag significantly increased the HN protein soluble overexpression. The expression level was the highest. The results from Western blotting and indirect ELISA showed that the purified HN protein had a good immunogenicity. Conclusion MBP tag enhances the expression and solubility of HN protein. The study will lay foundation for the research on the NDV mechanism, vaccine and diagnostic reagents.

Key words: Newcastle disease virus, Hemagglutinin-neuraminidase, Fusion tag, Flexible joint, Western blotting, Indirect ELISA, Chicken

张伟强郭豫杰李赛赛王月影陈丽颖杨国宇. 新城疫病毒血凝素-神经氨酸酶蛋白的可溶性超表达与免疫活性检测[J]. 解剖学报, 2016, 47(6): 848-855.

ZHANG Wei-qiang GUO Yu-jie LI Sai-sai WANG Yue-ying CHEN Li-ying YANG Guo-yu. Soluble overexpression and immunological activity of the Newcastle disease virus hemagglutinin-neuraminidase protein[J]. Acta Anatomica Sinica, 2016, 47(6): 848-855.

参考文献

［1］Dai CX, Kang H, Yang WQ, et al. O-2’-Hydroxypropyltrimethy ammonium chloride chitosan nanoparticles for the delivery of live Newcastle disease vaccine［J］. Carbohydr Polym, 2015, 130: 280-289.

［2］Absalon AE, Mariano-Matias A, Garcia LJ, et al. Complete genome analysis of velogenic Newcastle disease virus reference strain “Chimalhuacan”: evolution of viral lineages in Mexico［J］. Virus Genes, 2014, 49(2): 233-236.

［3］Dong DX, Gao J, Sun Y, et al. Adenovirus-mediated co-expression of the TRAIL and HN genes inhibits growth and induces apoptosis in Marek’s disease tumor cell line MSB1［J］. Cancer Cell Int, 2015, 15:20.

［4］Sun CX, Wen HL, Chen Y, et al. Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process［J］. Biosci Trends, 2015, 9(1): 56-64.

［5］Selvan N, Ranjit S Amirtha J, et al. Production and application of recombinant haemagglutinin neuraminidase of Newcastle disease virus［J］. Asian Pacific Journal of Tropical Medicine, 2010, 3(8): 629-632.

［6］Motamedi MJ, Amani J, Shahsavandi S, et al. In silico design of multimeric HNF antigen as a highly immunogenic peptide vaccine against newcastle disease virus［J］. International Journal of Peptide Research and Therapeutics, 2014, 20(20): 179-194.

［7］Li T, Wang GL, Shi BT, et al. Comprehensive analysis and characterization of linear antigenic domains on HN protein from genotype Ⅶ Newcastle disease virus using yeast surface display system［J］. PLoS One, 2015, 10(6): 1-14.

［8］Wang YH, Xue F, Zhu YM, et al. Charncterization of truncated glycoprotein D of infectious bovine reinotracheitis virus expressed in E.coli［J］. Chinese Journal of Preventive Veterinary Medicine, 2007, 29(11): 865-869.(in Chinese)

王延辉, 薛飞, 朱元茂, 等. 牛传染性鼻气管炎病毒gD蛋白的截短表达与活性检测［J］. 中国预防兽医学报, 2007, 29(11): 865-869.

［9］Tian LL, Li L. Development and application of indirect ELISA for detecting antibodies against newcastle disease virus with recombinant truncated HN protein expressed inE.coli［J］. China Poultry, 2012, 34(6): 29-32. (in Chinese)

田莉莉, 李丽. 新城疫病毒HN基因的截短表达及间接ELISA抗体检测方法的建立与应用［J］. 中国家禽, 2012, 34(6): 29-32.

［10］Vu TT, Jeong B, Yu J, et al. Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag［J］. Toxicon, 2014, 92: 157-165.

［11］Vu TT, Koo BK, Song JA, et al. Soluble overexpression and purification of bioactive human CCL2 in E-coli by maltosebinding protein［J］. Mol Biol Rep, 2015, 42(3): 651-663.

［12］Yang F, Pan YY, Chen Y, et al. Expression and purification of Canis interferon alpha in Escherichia coli using different tags［J］. Protein Expr Purif, 2015, 115: 76-82.

［13］Boubakri H, Seghezzi N, Duchateau M, et al. The absence of pupylation (Prokaryotic Ubiquitin-Like Protein Modification) affects morphological and physiological differentiation in streptomyces coelicolor［J］. J Bacterioly, 2015, 197(21): 3388-3399.

［14］Raran-Kurussi S, Keefe K, Wauqh DS. Positional effects of fusion partners on the yield and solubility of MBP fusion proteins［J］. Protein Expre Purif, 2015, 110: 159-164.

［15］Li XM, Song SY, Pei YX, et al. Oriented and reversible immobilization of His-tagged proteins on two-and three-dimensional surfaces for study of protein-protein interactions by a QCM biosensor［J］. Sensors and Actuators B Chemical, 2016, 224: 814-822.

［16］Hojin M, Joungeun P, Hyeon Y, et al. Development of a novel recombinant heamagglutinin-neuramindase Elisa (rHN-ELISA) for evaluation of humoral immunity in chicken vaccinated against Newcastle disease virus (NDV)［J］. Journal of Animal and Veterinary Advances, 2010, 9(23): 2932-2939.

［17］Yang X, Zhou YS, Li JN, et al. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge［J］. Arch Virol, 2016, 161(5): 1209-1216.

［18］Ran XH, Wen XB, Zhao XH. Expression of F and HN protein of NDV in E.coli［J］. Biotechnology, 2008, 18(3): 11-14. (in Chinese)

冉旭华, 闻晓波, 赵喜红. 新城疫病毒F和HN蛋白的原核表达［J］. 生物技术, 2008, 18(3): 11-14.

［19］Liu YH, Ding Zh, Chang Sh. Genetic variation and prokaryotic expression of HN gene of duck paramyxovirus［J］. Chinese Journal of Veterinary Science, 2007, 27(3): 315-318. (in Chinese)

刘艳华, 丁壮, 常爽. 血清Ⅰ型鸭源副粘病毒HN蛋白基因的遗传变异及原核表达［J］. 中国兽医学报, 2007, 27(3): 315-318.

［20］Luo JL, Qi Y, Ren T. Prokaryotic expression of antigen sites of the Newcastle disease virus hemagglutinin-neuraminidase［J］. Journal of South China Agricultural University, 2009, 30(4): 90-93. (in Chinese)

罗晶璐, 齐岩, 任涛. 新城疫病毒HN基因主要抗原位点区段的原核表达［J］. 华南农业大学学报, 2009, 30(4): 90-93.

［21］Zhu YP, Tian XL, Li P. Prokaryotic expression of the main antigen region in HN gene of newcastle disease virus xx08 strain［J］. He’nan Agricultural Sciences, 2012, 41(7): 134-137,154. (in chinese)

朱艳平, 田献礼, 李鹏. 新城疫病毒xx08毒株血凝素神经氨酸酶基因主要抗原区原核表达及鉴定［J］. 河南农业科学, 2012, 41(7): 134-137, 154.

［22］Geng G. Prokaryotic expression of the main antigen region in HN gene of newcastle disease virus ［J］. Animal Husbandry and Veterinary Medicine, 2013, 45(5): 53-56. (in chinese)

耿岗. 新城疫病毒HN蛋白主要抗原区域的表达与活性检测［J］. 畜牧与兽医, 2013, 45(5): 53-56.

［23］Wang B, Liu PX, Li T. Prokaryotic expression and functional analysis of the single chain antibody against the HN protein of Newcastle disease virus［J］. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(5): 383-386. (in chinese)

王斌, 刘培欣, 李涛. 抗新城疫病毒HN蛋白单链抗体的原核表达及其功能鉴定［J］. 中国预防兽医学报, 2014, 36(5): 383-386.

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