人表皮生长因子融合蛋白在干酪乳杆菌中的表达
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中央高校基本科研业务费专项(2662015PY038)


Expression of human epidermal growth factor fusion protein in Lactobacillus casei
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    摘要:

    以干酪乳杆菌作为传递载体,构建表达人表皮生长因子的重组干酪乳杆菌,探讨原位表达特定蛋白的可行性。采用SOE PCR合成人表皮生长因子序列,克隆到干酪乳杆菌表达载体pELWH,构建pELWH-hEGF重组质粒,将该质粒电转化干酪乳杆菌宿主,采用Western blot及间接免疫荧光检测目的蛋白的表达,并通过细胞增殖实验验证目的蛋白的生物学活性。结果表明:在重组菌的细胞表面及培养液上清中均检测到SlpA-hEGF融合蛋白,分子质量约55 ku;细胞增殖实验显示SlpA-hEGF融合蛋白及干酪乳杆菌上清均能显著促进NIH/3T3细胞的增殖。

    Abstract:

    Lactobacillus casei is a probiotic that can colonize in animal intestines.The recombinant L.casei was constructed to express human epidermal growth factor (hEGF) and to test if the bacterium has the potential of being used as live bacterial vector to express specific protein in situ to stimulate mucosal immunity.The hEGF was synthesized through splicing overlapping extension PCR (SOE PCR),and cloned into the LAB surface exhibition vector pELWH,yielding pELWH-hEGF.The plasmid resulted was electroporated into L.casei MCJΔ1.Transformants were then analyzed by Western blot and indirect immunofluorescent assay.Cell proliferation assay was used to investigate the bioactivity of hEGF expressed in L.casei.SlpA-hEGF fusion protein on the cell surface and in the supernatant was detected.The molecular weight of the SlpA-hEGF fusion protein was estimated to be approximately 55 ku.The result of cell proliferation assay showed that the SlpA-hEGF fusion protein and the supernatant can stimulates the proliferation of NIH/3T3 cells dramatically.It will lay the foundation for L.casei having the potential of being an excellent live vector for protein delivery.

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汪舒庆,葛向阳,陈正军.人表皮生长因子融合蛋白在干酪乳杆菌中的表达[J].华中农业大学学报,2018,37(04):70-77

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  • 收稿日期:2017-05-09
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  • 在线发布日期: 2018-07-09
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