长双歧杆菌C16 bif黏附基因敲除菌株的构建
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黑龙江省自然科学基金杰出青年项目 (JJ2020JQ0091)


Construction of Bifidobacterium longum C16 bif Adhesion Gene Knockout Strain
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    摘要:

    长双歧杆菌是定植于人体肠道中的优势菌株,与人体肠道健康密切相关。黏附是长双歧杆菌定植于肠道的前提,研究长双歧杆菌的黏附机制尤为重要。bif黏附基因作为长双歧杆菌的一类胞外黏附因子,其敲除后黏附效力是否丧失尚未明确。本试验利用同源重组技术,以长双歧杆菌为研究对象,构建bif黏附基因敲除突变株,验证该基因的黏附作用。首先以长双歧杆菌C16基因中的bif黏附基因和PBR322为模版,扩增bif黏附基因上、下游同源臂和tet抗性基因,然后分别将它们连接到pMD19-t simple Vecter上,对中间载体pMD19-up、pMD19-down、pMD19-tet进行双酶切,并将回收片段连接到PUC18上构成敲除载体PUC18-up-tet-down。通过电转化的方式导入宿主菌株中,利用同源重组筛选获得长双歧杆菌bif黏附基因缺失菌株。菌落PCR和测序结果均显示bif黏附基因敲除菌株构建成功,为进一步研究长双歧杆菌bif基因的黏附作用奠定基础。

    Abstract:

    Bifidobacterium longum is a dominant strain colonized in the human intestine and is closely related to human intestinal health. Adhesion is a prerequisite for colonization of Bifidobacterium longum in the intestine, so it is particularly important to study the adhesion mechanism of Bifidobacterium longum. bif is a type of extracellular adhesion factor, but its specific adhesion in the intestine has not been clarified. In this experiment, homologous recombination technology was used to construct bif adhesion knockout mutants to study the adhesion of bif gene. Firstly, the bif adhesion gene and PBR322 in the C16 gene of Bifidobacterium longum were used as templates to amplify the upstream and downstream homology arms and tet resistance genes of the bif adhesion gene, and then the upstream and downstream homology arms and tet of the bif adhesion gene, respectively. The resistance gene was ligated to pMD19-t simple vecter, and the intermediate vector pMD19-up, pMD19-down, pMD19-tet was double-digested, and the recovered fragment was ligated to PUC18 to form the knockout vector PUC18-up-tet-down. The host strain was introduced into the strain by electroporation, and the Bifidobacterium longum bif adhesion gene-deficient strain was obtained by homologous recombination screening. The results of colony PCR and sequencing showed that the construction of bif adhesion gene knockout strain was successful, which laid a foundation for further study on the adhesion of bifidobacteria bif gene.

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谭颖;陈晓倩;付恒芳;朱旭萌;李春;刘丽波.长双歧杆菌C16 bif黏附基因敲除菌株的构建[J].中国食品学报,2021,21(7):52-59

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  • 在线发布日期: 2021-08-12
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