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俞小琴, 魏玲, 刘秋英等. 人VIGILIN基因分段克隆及表达[J]. 四川大学学报（医学版）, 2015, 46(5): 661-666.
引用本文: 俞小琴, 魏玲, 刘秋英等. 人VIGILIN基因分段克隆及表达[J]. 四川大学学报（医学版）, 2015, 46(5): 661-666.
shu
YU Xiao-qin, WEI Ling, LIU Qiu-ying. et al. Segment Cloning and Expression of Human VIGILIN Gene[J]. Journal of Sichuan University (Medical Sciences), 2015, 46(5): 661-666.
Citation: YU Xiao-qin, WEI Ling, LIU Qiu-ying. et al. Segment Cloning and Expression of Human VIGILIN Gene[J]. Journal of Sichuan University (Medical Sciences), 2015, 46(5): 661-666.
shu

人VIGILIN基因分段克隆及表达

Segment Cloning and Expression of Human VIGILIN Gene

    摘要
  • 摘要: 目的 探讨人高密度脂蛋白结合蛋白(HDLBP)-VIGILIN蛋白与其他蛋白质相互作用的机制。 方法 根据 VIGILIN基因全长编码区的结构域，将 VIGILIN基因全长编码区分为N端、KH1-7、KH8-12、KH13-14、C端5段，以pDsred2-N1/ VIGILIN为模板分别扩增5个片段及全长cDNA后克隆至pGEX 5X 3原核表达载体, 测序,然后将鉴定后的重组质粒转化到表达宿主菌E.coli BL21中进行诱导表达GST-VIGILIN融合蛋白,并用SDS-PAGE电泳及Western blot检测蛋白表达结果。结果 成功扩增了人 VIGILIN基因编码区分段片段，并且构建到原核表达载体中，经酶切、测序鉴定证实序列完全正确，重组载体构建成功，并且在pGEX原核表达系统中诱导表达GST-VIGILIN融合蛋白，经SDS-PAGE电泳及Western blot检测初步证实表达成功。 结论 本实验首次根据VIGILIN蛋白不同的结构域成功构建了GST-VIGILIN分段克隆原核表达载体，并且对融合蛋白表达条件进行了优化,成功表达了GST 融合蛋白。

     

    Abstract: Objective To investigate the mechanisms of interaction between high-density lipoprotein binding protein (HDLBP)-VIGILIN with other proteins, we cloned VIGILIN cDNA N, KH1-7, KH8-12, KH13-14, and C fragments separately into expression vector, and identify the expressed proteins. Methods The recombinant plasmid pDsred2-N1/ VIGILIN was used as template to amplify VIGILIN full length, VIGILIN Nterminal, KH1-7, KH8-12, KH13-14, C terminal and recombinated them with pGEX 5X 3. After transformed into E.coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, we induced GST-VIGILIN fusion proteins on the appropriate conditions. Results The recombinant plasmids of pGEX 5X 3/ VIGILIN FL, pGEX 5X 3/ VIGILIN N terminal, pGEX 5X 3/ VIGILIN KH1-7, pGEX 5X 3/ VIGILIN KH8-12, pGEX 5X 3/ VIGILIN KH13-14, pGEX 5X 3/ VIGILIN C terminal were constructed successfully, and induced the GST-VIGILIN fusion proteins. Conclusion pGEX 5X 3/ VIGILIN FL, pGEX 5X 3/ VIGILIN N terminal, pGEX 5X 3/ VIGILIN KH1-7, pGEX 5X 3/ VIGILIN KH8-12, pGEX 5X 3/ VIGILIN KH13-14, pGEX 5X 3/ VIGILIN C terminal recombinant plasmids were constructed successfully, and their corresponding fusion proteins were successfully expressed.

     

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