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    • 不同实验条件对淋病奈瑟球菌体外转化效率的影响

    Li Xiaodie,Zhu Lin,Hong Yiyong,Huang Ying,Lai Wei,Gong Zijian

    Chinese Journal of Dermatology（2024）

    Third Affiliated Hospital of Sun Yat-sen University

    Cited 0|Views4
    Abstract
    目的 探讨不同实验条件对淋病奈瑟球菌(淋球菌)体外转化效率的影响.方法 以淋球菌D株DNA为模板扩增penA基因,采用重叠延伸PCR制备penAA501V(耐药突变)基因,购买淋球菌penAH041基因(耐药阳性对照).制备含penA/penAA501V/penAH041基因的质粒转化至大肠杆菌DH5α感受态细胞,采用蓝白筛选培养基克隆扩增;分别采用日本TaKaRa公司质粒提取试剂盒和天根生化科技(北京)有限公司高纯度质粒小提试剂盒提取质粒.以penA质粒、penAA501V质粒、penAH041质粒、penAA501V基因、penAH041基因为底物采用直接孵育法转化淋球菌受体菌株A43、A49,测定转化率(CFU/µg)及头孢曲松(CRO)的最小抑菌浓度(MIC).以penAA501V、penAH041基因为底物,采用脂质体法转化受体菌株A43、A49,观察转化效率及头孢曲松的MIC.结果 以D株淋球菌DNA为模板,成功获得1 749 bp的penA及penAA501V基因.提取质粒并电泳后,TaKaRa试剂盒提取的含淋球菌penA、penAA501V、penAH041基因的质粒主要集中在2 500 bp处,迁移速度较快;天根试剂盒提取的质粒主要集中在5 000 bp处,迁移速度较慢.直接孵育法转化时,含野生型penA的A43株的MIC为0.001 μg/ml,转入penA质粒后MIC上升至0.002 μg/ml,转入penAA501V质粒和penAA501V基因转化株MIC均为0.004 μg/ml,转入阳性对照penAH041质粒和penAH041基因后MIC均升至0.512 μg/ml;各底物孵育后CRO对A49株的MIC值与空白对照相同,均为0.016 μg/ml.脂质体法转化时,含野生型penA的A43 株对 CRO 的 MIC 是 0.002 μg/ml,A49 株 MIC 为 0.016 μg/ml;penAA501V 基因转化 A43 株后 MIC 升至0.004 μg/ml,转化A49株后仍为0.016 μg/ml;阳性对照penAH041基因转化A43和A49株后MIC均升至0.250 μg/ml,较空白对照分别升高至125倍和15.6倍.结论 选择质粒为底物采用直接孵育法对A43株淋球菌有较好转化效率,不同质粒提取方法可能影响转化效率,对于直接孵育法转化效率不高的A49株可采用脂质体法.
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    Neisseria gonorrhoeae,Transformation,bacterial,Plasmids,Ceftriaxone,Drug resistance,penA gene,Minimum inhibitory concentration
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    【要点】：研究不同实验条件对奈瑟菌体外转化效率的影响，发现使用质粒为底物和直接共培养法对菌株A43有更高的转化效率，而脂质体法适用于转化效率较低的菌株A49。
    

    【方法】：通过重叠延伸PCR法准备耐药性penAA501V基因，购买耐药性penAH041基因作为阳性对照，使用两种不同的质粒提取试剂盒提取质粒，并通过直接共培养法和脂质体法将基因转化到奈瑟菌受体菌株A43和A49中。
    

    【实验】：使用菌株D的基因组DNA为模板成功扩增出penA和penAA501V基因，通过电泳分析提取的质粒，发现不同提取方法对质粒迁移率有影响。直接共培养法转化后，菌株A43的CRO最小抑菌浓度(MIC)从野生型的0.001 μg/ml上升至不同水平；菌株A49的MIC在转化后保持不变。脂质体法转化后，菌株A43和A49的MIC均有上升，尤其是阳性对照penAH041基因转化后上升更为显著。