[1]孙守勋,刘 静,解立威,等.人TLR2胞外区基因酵母表达载体的构建及表达研究[J].现代检验医学杂志,2018,33(03):24-26，30.[doi:10.3969/j.issn.1671-7414.2018.03.008]
　SUN Shou-xun,LIU Jing,XIE Li-wei,et al.Construction and Expression of Expressive Vector of the TLR2 Extracellular Domain Gene of Human in Yeast Cell[J].Journal of Modern Laboratory Medicine,2018,33(03):24-26，30.[doi:10.3969/j.issn.1671-7414.2018.03.008]
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人TLR2胞外区基因酵母表达载体的构建及表达研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第33卷
期数:
2018年03期
页码:
24-26，30
栏目:
论著
出版日期:
2018-07-17

文章信息/Info

Title:
Construction and Expression of Expressive Vector of the TLR2 Extracellular Domain Gene of Human in Yeast Cell
文章编号:
1671-7414(2018)03-024-04
作者:
孙守勋1; 刘 静2; 解立威1; 柏雪婷1; 单 娜1; 孟冬娅1
1.中一东北国际医院临床检验中心,沈阳 110179; 2.沈阳军区总医院检验科,沈阳 110003
Author(s):
SUN Shou-xun1; LIU Jing2; XIE Li-wei1; BAI Xue-ting1; SHAN Na1; MENG Dong-ya1
1.Department of Central Clinical Laboratory, Northeast International Hospital,Shenyang 110179,China; 2.Department of Clinical Laboratory,General Hospital of PLA,Shenyang 110003,China
关键词:
人Toll样受体2;  基因表达;  酵母双杂交
分类号:
Q786
DOI:
10.3969/j.issn.1671-7414.2018.03.008
文献标志码:
A
摘要:
目的 在真核生物酵母细胞中表达人Toll样受体2(TLR2)胞外区基因。方法 以重组质粒pAdTrack-TLR2为模板,应用PCR方法扩增TLR2胞外区基因,克隆到pMD18-T载体中并测序鉴定,酶切回收后连接到酵母表达质粒pGBKT7中并转化酵母菌Y187,测定其在Y187中的自激活现象及毒性,提取酵母蛋白质,行SDS-PAGE电泳和Western blot分析。结果 成功扩增出TLR2胞外区基因,测序结果与GenBank中序列一致。酶切回收的目的基因成功克隆入酵母表达载体pGBKT7中并转化酵母菌Y187后证实无重组质粒的自激活作用,Western blot分析显示该基因在酵母细胞中表达。结论 成功构建了TLR2胞外区基因的酵母表达载体,并在酵母细胞Y187中正确表达,为后续研究奠定了基础。
Abstract:
Abstract:Objective To construct and express the TLR2 extracellular domaingene of human in yeast cell.Methods Human TLR2 extracellular domain cDNA was amplified by PCR from recombinant plasmids pAdTrack-TLR2 and inserted into pMD18-T vector.The gene of TLR2 was cut from pMD18-TLR2 vector and cloned into yeast expressive plasmid pGBKT7,and pGBKT7-TLR2 was then transformed into yeast Y187.The toxicity and self-activationof the pGBKT7-TLR2 in Y187 yeast was tested.The yeast protein was isolated and analyzed with SDS-PAGE and Western blotting hybridization.ResultsThe gene fragment was successfully obtained by PCR. After TA cloned,its sequence was in conformity with the sequence reported in GenBank.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell Y187,and no self-activating transcriptional activation and toxicity was observedin Y187.The SDS-PAGE and Western blotting assay showed that the TLR2 protein existed in yeast cells.Conclusion The TLR2 extracellulardomain gene of human was successfully expressed into yeast system,which lays the foundation for further study on the structure and biological activity of TLR2.

参考文献/References:

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备注/Memo

备注/Memo:
作者简介:孙守勋(1979-),男,医学硕士,副主任技师,主要从事微生物学与分子生物学方面的研究,E-mail:sunshouxun_79@163.com。 通讯作者:孟冬娅,E-mail:13309889399@189.cn。
更新日期/Last Update: 2018-04-16