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- 2017
猪传染性胃肠炎病毒M基因酵母双杂交诱饵载体的构建及鉴定
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Abstract:
筛选与猪传染性胃肠炎病毒(TGEV)M蛋白相互作用的宿主蛋白，构建M蛋白的诱饵载体pGBKT7-M.首先利用PCR技术从重组表达载体pFastBacTM-M中扩增得到M基因，定向克隆至载体pMD19-T simple，验证正确后克隆至酵母双杂交系统诱饵表达载体pGBKT7，经双酶切、PCR反应及测序验证其正确插入后，利用PEG/LiAc法将构建好的重组诱饵载体pGBKT7-M及空载体pGBKT7分别转化到酵母Y2HGold感受态细胞中，检测其对酵母菌株是否有毒性作用和自激活活性及诱饵蛋白在酵母细胞内的表达情况.结果表明：扩增得到M基因738 bp，成功构建了诱饵表达载体pGBKT7-M，此诱饵载体对酵母细胞无细胞毒性和自激活活性. Western blot检测到5×104左右的蛋白条带，显示诱饵蛋白在酵母细胞内稳定表达.
A bait vector for M protein (pGBKT7-M) was constructed for screening the host proteins of the interactions between TGEV and M protein. First, the gene from the structural proteins M was amplified from pFastBacTM-M by PCR and cloned into the vector pMD19-T simple. After being verified, it was directly cloned into the bait vector pGBKT7 of the yeast two-hybrid system. Then the recombinant plasmid was identified by PCR, restriction enzyme digestion and sequence analysis and transformed into the yeast cells Y2HGold by the PEG/LiAc method. Toxicity and self-activation of the bait protein were detected using different droupout minimal bases, and the bait vector pGBKT7-M was shown to have no toxicity to the yeast cells and no self-activation phenomenon to the report genes. The above results indicated that the constructed bait vector pGBKT7-M can be used for screening the host proteins interacting with TGEV M protein using the yeast two-hybrid system
| [1] | ITO T, CHIBA T, OZAWA R, et al. A Comprehensive Two-Hybrid Analysis to Explore the Yeast Protein Interactome[J]. Proceedings of the National Academy of Sciences, 2001, 98(8): 4569-4574. DOI:10.1073/pnas.061034498 |
| [2] | SHI Y H, CHEN J, LI C H, et al. The Establishment of a Library Screening Method Based on Yeast Two-Hybrid System and Its Use to Determine the Potential Interactions of Liver Proteins in Ayu, Plecoglossus Altivelis[J]. Fish Shellfish Immunol, 2011, 30(4): 1184-1187. |
| [3] | LAMPHEAR B J, JILKA J L, KESL L, et al. A Corn-Based Delivery System for Animal Vaccines: an Oral Transmissible Gastroenteritis Virus Vaccine Boosts Lactogenic Immunity in Swine[J]. Vaccine, 2004, 22(19): 2420-2424. DOI:10.1016/j.vaccine.2003.11.066 |
| [4] | ZOU H, ZARLENGA D S, SESTAK K, et al. Transmissible Gastroenteritis Virus: Identification of M Protein-Binding Peptide Ligands with Antiviral and Diagnostic Potential[J]. Antiviral Research, 2013, 99(3): 383-390. DOI:10.1016/j.antiviral.2013.06.015 |
| [5] | SCHWEGMANN-WESSELS C, HERRLER G. Transmissible Gastroenteritis Virus Infection: a Vanishing Specter[J]. Dtsch Tierarztl Wochenschr, 2006, 113(4): 157-159. |
| [6] | de HAAN C A, VENNMAE H, ROTTIERO P J. Assembly of the Coronavirus Envelope: Homotypic Interactions between the M Proteins[J]. Journal of Virology, 2000, 74(11): 4967-4978. DOI:10.1128/JVI.74.11.4967-4978.2000 |
| [7] | GAROFF H, HEWSON R, OPSTELTEN D J. Virus Maturation by Budding[J]. Mirobiol Mol Biol Rev, 1998, 62(4): 1171-1190. |
| [8] | WANG Ji-bin, FANG Shou-guo, XIAO Han, et al. Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and its Implication in Virion Assembly and Budding[J]. Plos One, 2009, 4(3): e4908. DOI:10.1371/journal.pone.0004908 |
| [9] | LIN K P, LIU D X. The Missing Link in Coronavirus Assembly[J]. J Biol Chem, 2001, 276(3): 17515-17523. |
| [10] | SPAAN W, CAVANAGH D, HORZINEK M C. Coronaviruses: Structure and Genome Expression[J]. J Gen Virol, 1988, 69(12): 2939-2952. DOI:10.1099/0022-1317-69-12-2939 |
| [11] | KLUMPERMAN J, LOCKER J K, MEIJER A, et al. Coronavirus M Proteins Accumulate in the Golgi Complex beyond the Site of Virion Budding[J]. Journal of Virology, 1994, 68(10): 6523-6534. |
| [12] | ESCORS D, ORTEGO J, LAUDE H, et al. The Membrane M Protein Carboxy Terminal Binds to Transmissible Gastroenteritis Coronavirus Core and Contribute to Core Stability[J]. Journal of Virology, 2001, 75(3): 1312-1324. DOI:10.1128/JVI.75.3.1312-1324.2001 |
| [13] | MEGAN M S, DARLA D, ERIC H, et al. Detecting Protein-Protein Interactions in Vesicular Stomatitis Virus Using a Cytoplasmic Yeast Two Hybrid System[J]. Journal of Virological Methods, 2011, 173(2): 203-212. DOI:10.1016/j.jviromet.2011.02.006 |
| [14] | FIELD S, SONG O. A Novel Genetic System to Detect Protein-Protein Interaction[J]. Nature, 1989, 340(6230): 245-246. DOI:10.1038/340245a0 |
| [15] | 殷震, 刘景华. 动物病毒学[M]. 2版. 北京: 科学出版社, 1997. |
| [16] | 王华, 孙成友, 王淑娟, 等. 蛋白组学在病毒致病机理研究中的应用[J]. 现代生物医学进展, 2014, 14(13): 2583-2585. |
| [17] | 蒙学莲, 朱学亮, 翟军军, 等. 小反刍兽疫病毒酵母双杂交诱饵载体pGBKT7-tH的构建、表达和鉴定[J]. 中国兽医学报, 2011, 31(4): 509-512. |
| [18] | DONG Bo, GAO Wei, LU Hui-jun, et al. A Small Region of Porcine Hemagglutinating Encephalomyelitis Virus Spike Protein Interacts with the Neural Cell Adhesion Molecule[J]. Intervirology, 2015, 58(2): 130-137. DOI:10.1159/000381060 |
| [19] | GISSEN P, MAHER E R. Cargos and Genes: Insights into Vesicular Transport from Inherited Human Disease[J]. Journal of Medical Genetics, 2007, 44(9): 545-555. DOI:10.1136/jmg.2007.050294 |
| [20] | GRIFFITHS G, ROTTIER P. Cell Biology of Viruses that Assemble along the Biosynthetic Pathway[J]. Semin Cell Biol, 1992, 3(5): 367-381. DOI:10.1016/1043-4682(92)90022-N |
| [21] | STURMAN L S, HOLMES K V, BEHNKE J. Isolation of Coronavirus Envelope Glycoproteins and Interaction with the Nucleocapsid[J]. J Virol, 1980, 33(1): 449-462. |
| [22] | SIDDLE S G, ANDERSON R, CAVANGNAGH D, et al. Coronaviridae[J]. Intervirology, 1983, 20(4): 181-189. DOI:10.1159/000149390 |
| [23] | 韩国全, 郭万柱, 赖维莉, 等. 猪传染性胃肠炎病毒四川株M蛋白基因克隆分析及真核表达载体构建[J]. 黑龙江畜牧兽医, 2007, 01: 66-68. DOI:10.3969/j.issn.1004-7034.2007.01.034 |
