VSV糖蛋白酵母双杂交诱饵载体的构建及其自激活作用的检测

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 109-114.

VSV糖蛋白酵母双杂交诱饵载体的构建及其自激活作用的检测

韩岩岩¹, 宋德光²

(1.遵义医学院公共卫生学院，遵义563003；
2. 吉林大学畜牧兽医学院，长春 130062)

收稿日期:2013-12-28 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:韩岩岩，女，博士，研究方向：分子营养学和营养相关疾病；E-mail：hanyanyan1984@126.com
基金资助:
贵州省科学技术基金项目(黔科合J字［2013]2313号)，遵义医学院博士启动基金项目(F-584)

Construction of a Bait Vector for VSV Glycoprotein and Evaluation of Its Self-activation in Yeast Two-hybrid System

Han Yanyan¹, Song Deguang²,

(1. School of Public Health，Zunyi Medical University，Zunyi 563003；
2. College of Animal science and Veterinary Medicine，Jilin University，Changchun 130062)

Received:2013-12-28 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 构建水泡性口炎病毒糖蛋白(VSV-G)酵母双杂交诱饵载体，检测其在酵母细胞中的表达和自激活作用，为进一步研究酵母双杂交系统筛选与VSV-G相互作用蛋白奠定基础。通过RT-PCR方法从水泡性口炎病毒(VSV)克隆糖蛋白(G)基因，BamH I和Sal I双酶切后连接诱饵载体pSos，获得诱饵质粒pSos-VSV-G，经测序鉴定后pSos-VSV-G转化到酵母菌株cdcH25(α)，在营养缺陷培养基中观察pSos-VSV-G的自激活作用和毒性作用，同时利用蛋白印迹法分析诱饵蛋白的表达。成功扩增VSV-G基因，并准确克隆入pSos中，诱饵载体pSos-VSV-G成功转化到酵母菌株cdcH25(α)中，经表型筛选检测无自激活作用和毒性作用，蛋白印迹法检测证实pSos-VSV-G在酵母细胞中表达诱饵蛋白。可以利用酵母双杂交系统筛选与VSV-G相互作的蛋白质。

关键词: 水泡性口炎病毒, 糖蛋白, 酵母双杂交, 诱饵载体

Abstract: It was to construct a bait vector for vesicular stomatitis virus glycoprotein(VSV-G)and detect its protein expression and self-activation activity in yeast cells, as a basis for further study of screening and VSV-G protein interaction in two hybrid system. Fragment of VSV-G gene were amplified using RT-PCR and directly ligated to pSos vector, insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The self-activation and toxicity of the bait plasmid transformed into the yeast cells cdcH25(α)was observed in selective culture medium, and the bait protein was confirmed by Western blot. Results showed that VSV-G gene was found in the reconstructed plasmid pSos-VSV-G by sequencing. Yeast two-hybrid tests showed that yeast cells cdcH25(α)transfected with pSos showed that VSV-G had no self-activation and toxicity, the expression of bait protein was confirmed by Western blot. The yeast two-hybrid system can be applied to screen the interacting proteins of VSV-G.

Key words: Vesicular stomatitis virus, Glycoprotein, Yeast two-hybrid, Bait plasmid

韩岩岩, 宋德光. VSV糖蛋白酵母双杂交诱饵载体的构建及其自激活作用的检测[J]. 生物技术通报, 2014, 0(4): 109-114.

Han Yanyan, Song Deguang,. Construction of a Bait Vector for VSV Glycoprotein and Evaluation of Its Self-activation in Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2014, 0(4): 109-114.

参考文献

[1] 殷震, 刘景华. 动物病毒学［M]. 第2版, 北京：科学出版社. 1997：799-805.
[2] 马俊儒, 马相斋. 牛暴发水疱性口炎[J]. 畜牧与兽医, 2003, 35(1)：44-48.
[3] Jayakar HR, Whitt MA. Identification of two additional translation products from the matrix(M)gene than contribute to VSV cytopathology[J]. J Virol, 2002, 76(16)：8011-8018.
[4] Roberts PC, Kipperman T, Compans RW. Vesicular stomatitis virus g protein acquires pH-independent fusion activity during transport in a polarized endometrial cell line[J]. Virol Dec, 1999；73：10447 - 10457.
[5] Coil DA, Miller AD. Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus[J]. J Virol, 2004, 78(20)：10920-10926.
[6] Rose JK, Gallione CJ. Nucleotide sequences of the mRNA’s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions[J]. J Virol, 1981, 39：519-528.
[7] Feilotter HE, Hannon GJ, Ruddell CJ, et al. Construction of an improved host strain for two hybrid screening[J]. Nucl Acids Res, 1994, 22：1502-1506.
[8] Tian QY, Zhao YP, Liu CJ. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin[J]. J Vis Exp, 2012, 17(59)：3562.
[9] Barak, Huang W, Wang SL, Lozano G, et al. cDNA library screening using the SOS recruitment system[J]. Biotechniques, 2001, 30(1)：94-99.
[10] Gietz RD, Schiestl RH, Williems AR, et al.Studies on the transfor-mation of intact yeast cells by the LiAc/SS-DNA/PEG procedure[J]. Yeast, 1995, 11(4)：355-360.
[11] Serebriiskii IG, Kotova E. Analysis of protein-protein interactions utilizing dual bait yeast two-hybrid system[J]. Method Mol Biol, 2004, 261：263-296.
[12] Uetz P, Glot L, Cagney G, et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae[J]. Nature, 2000, 403：623-627.

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