CN111575302A - 一种结核分枝杆菌的突变基因及其应用 - Google Patents
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Abstract
本发明属于生物医药的技术领域，本发明提供了一种结核分枝杆菌的突变基因及其应用。所述突变基因是在结核分枝杆菌标准株H37Rv基因中缺失了基因片段Rv1527c的突变基因，或结核分枝杆菌的Rv1527c基因片段上第1380位的丙氨酸突变为脯氨酸或第1637位的甘氨酸突变为丙氨酸的突变基因。将上述突变基因作为结核分枝杆菌的生物标志物，用于结核病患者治疗前临床标本检测，有利于临床制定合理的化疗方案，从而提高临床结核病、尤其是耐多药结核病患者的治疗成功率。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种结核分枝杆菌的突变基因及其应用。
背景技术
氨基糖苷类是世界卫生组织推荐的注射用抗结核药物，包括链霉素(STR)、阿米卡星(AMI)和卡那霉素(KAN)。STR主要用于普通结核病的治疗，而AMI和KAN目前是治疗耐多药结核病(multidrug-resistant tuberculosis，MDR-TB，指结核分枝杆菌同时耐利福平和异烟肼两种抗结核药物)的核心二线药物。STR、AMI和KAN作用于结核分枝杆菌(Mtb)的核糖体，干扰细菌蛋白正常合成，在体外有较强的杀菌活性。目前已知rrs、rpsL、eis启动子和tlyA基因突变与结核分枝杆菌对氨基糖苷类耐药有关。
结核分枝杆菌对链霉素(STR)耐药主要与rrs和rpsl基因突变有关：rrs A514C突变可能与STR低度耐药相关；rpslK43R突变与STR高度耐药相关，rpslK88Q/R突变与STR中低度耐药相关。在不同的研究中，24％～89％链霉素表型耐药菌株携带了rpslK43R突变，5％～27％携带rpslK88Q/R突变。总体上，75％～90％的链霉素耐药菌中存在rrs或rpsl的相关耐药突变。结核分枝杆菌对卡那霉素(KAN)与阿米卡星(AMI)耐药主要与rrs和eis基因突变有关，研究显示，rrsA1408G与临床分离株的KAN高浓度耐药密切相关。56％的KAN耐药株携带了rrsA1408G突变；78％的AMI耐药株携带了rrs A1408G突变。eis突变与KAN和AMI的低浓度耐药相关，但该现象仅出现在体外试验中。在已发表的数据中，22％的KAN耐药株携带了eisA-10A突变，13％携带C-12T突变，11％携带了C-14T突变，5％携带了G-37T突变。结核分枝杆菌对STR、KAN、AMI三药均耐药的基因型改变是rrsA1401G突变联合rpsL突变，或者联合rrsA514C突变。
然而，结核分枝杆菌(Mtb)对氨基糖苷类抗结核药物之间存在交叉耐药性，抗生素使用不当可能导致治疗无效并且增加药物的不良反应。目前已有氨基糖苷类抗结核药物的分子药敏检测技术，针对rrs或rpsl基因突变检测，STR耐药菌可达75％～90％的检测率，但对AMI和KAN耐药菌的检测仅有5％～78％，敏感性和特异性均有待提高。且已有氨基糖苷类抗结核药物的分子药敏检测技术不能直接指导临床用药，因此难以提高治愈率。因此，深入了解结核分枝杆菌临床分离菌对氨基糖苷类药物的耐药性，有助于耐药的快速诊断，有利于临床制定合理的化疗方案。
发明内容
本发明的目的在于提供一种结核分枝杆菌的突变基因，并将该突变基因作为结核分枝杆菌的生物标志物。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种结核分枝杆菌的突变基因，所述突变基因是在结核分枝杆菌标准株H37Rv基因中缺失了基因片段Rv1527c的突变基因，或结核分枝杆菌的Rv1527c基因片段上第1380位的丙氨酸突变为脯氨酸或第1637位的甘氨酸突变为丙氨酸的突变基因。
优选的，所述基因片段Rv1527c(ID:886442)位于结核分枝杆菌标准株H37Rv基因中的第1722083～1728409位。
优选的，所述结核分枝杆菌标准株H37Rv的基因登录号为NC_000962.3。
本发明还提供了所述突变基因作为结核分枝杆菌生物标志物的应用。
本发明提供的突变基因对氨基糖苷类抗结核药物具有较强的敏感性和特异性，提示该突变基因对氨基糖苷类抗结核药物具有敏感性。将本发明提供的结核分枝杆菌的突变基因作为检测结核分枝杆菌的生物标志物，用于结核病患者治疗前临床标本检测，有利于临床制定合理的化疗方案，从而提高临床结核病、尤其是耐多药结核病患者的治疗成功率。
附图说明
图1普通PCR及RealTimePCR验证H37Rv△Rv1527c构建成功。
图2为H37Rv标准菌和H37RvΔRv1527c敲除菌对12种药物的MIC试验。
图3为一例耐多药肺结核患者二线抗结核药治疗前后胸部CT双肺病灶吸收情况。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
利用噬菌体介导的结核分枝杆菌基因敲除技术敲除实验室标准菌株H37Rv的Rv1527c基因片段，成功构建结核分枝杆菌Rv1527c基因敲除株H37RvΔRv1527c，进一步研究Rv1527c基因的功能。设计Rv1527c基因的左(L)、右(R)臂引物，即Rv1527c-LFP(5'-TTTTTTTTCCATAAATTGGTTCAGCGACGACGGCCT-3'),Rv1527c-LRP(5'-TTTTTTTTCCATTTCTTGGACCCCATACCAATGACAGCTA-3')和Rv1527c-RFP(5'-TTTTTTTTCCATAGATTGGATCCGCATTTCCGCCAC-3'),Rv1527c-RRP(5'-TTTTTTTTCCATCTTTTGGGGCGGTAGGCCCAGTTCA-3')，PCR扩增Rv1527c基因L、R臂，构建同源交换位点(AES)；随后将其整合到结核分枝杆菌噬菌体基因组，获得噬菌粒；将噬菌粒导入耻垢分枝杆菌，经体外扩增获得高滴度的重组噬菌体，对结核分枝杆菌进行转染并涂布到潮霉素抗性的固体培养基，挑取单克隆接种于含潮霉素抗性的液体培养基，37℃培养4～5周。提取H37Rv△Rv1527c全基因组，设计左/右段上/下游验证引物对(LYZFP/LYZRP、RYZFP/RYZRP)，即LYZFP(5'-CAATACAACTGGACCGGATGA-3')、LYZRP(5'-GTGGACCTCGACGACCCTAG-3')、RYZFP(5'-TGGATCTCTCCGGCTTCACC-3')、RYZRP(5'-CGGGTCGTAGGCTTGGATTT-3')。通过PCR检测，结果如图1，图1A表示普通PCR验证H37Rv△Rv1527c构建成功，Lane1和Lane3表示以H37Rv基因组为模板，分别使用LYZFP/LYZRP和RYZFP/RYZRP引物对呈现凝胶图结果；Lane2和Lane4表示以H37RvΔRv1527c基因组为模板，使用LYZFP/LYZRP和RYZFP/RYZRP所出现的凝胶图结果；M：DNAMarker。图1B表示RealTimePCR验证H37Rv△Rv1527c构建成功；用Graphpad prim6做统计学分析，采用t检验，*代表P<0.05，实验重复3次。验证H37RvΔRv1527c构建成功。
实施例2
针对目前常用的12种抗结核药物对结核分枝杆菌的H37Rv标准菌和H37Rv△Rv1527c敲除菌进行最低抑菌浓度(MIC)试验。
使用美国赛默飞公司生产的TREK
MYCOTB药敏板(简称“MYCOTB”)，含12种抗结核药物冻干粉的最低抑菌浓度(minimum inhibitory concentration,MIC)药敏板，药物包括氧氟沙星、莫西沙星、利福平、阿米卡星、链霉素、利福布丁、对氨基水杨酸、乙硫异烟胺、环丝氨酸、异烟肼、卡那霉素和乙胺丁醇。药敏板使用美国标准菌库(AmericanType Culture Collection，ATCC)H37Rv(ATCC 27294)和H37Ra(ATCC 25177)进行质量控制以确保药敏板上药物的有效活性。
结核分枝杆菌药敏检测的临界浓度参考美国临床和实验室标准协会(Clinicaland Laboratory Standards Institute，CLSI)M24-A2及食品药品监督管理局(Food andDrug Administration，FDA)批准的药敏实验解释标准；测试菌株对某药的MIC值小于或等于临界浓度，说明该菌株对该药临界浓度敏感，测试菌株对某药的MIC值大于临界浓度，说明该菌株对该药临界浓度表现为耐药。
分别制备0.5McFarland标准的H37Rv标准菌和H37Rv△Rv1527c敲除菌的菌悬液，分别取100μL菌悬液至11mL7H9+10％OADC药敏接种培养液涡旋振荡混匀30秒。在分别分装100uL菌悬液至96孔MYCOTB药敏板。并设只加菌液的阳性对照。完成后使用粘合密封膜覆盖上样完成的MYCOTB药敏板，放置37℃孵育箱静置培养，分别于10天、21天观察结果。
结果如图2所示：图2中A、C分别表示H37Rv标准菌的原始图及模拟图，B、D分别表示H37Rv△Rv1527c敲除菌的原始图及模拟图；具有下划线的数字为对应药物的临界浓度(μg/mL)；椭圆阴影标记提示MTB(结核分枝杆菌)生长(High→Low提示浓度由高到低)。12种抗结核药物分别为：OFL氧氟沙星，MXF莫西沙星，RIF利福平，AMI阿米卡星，STR链霉素，RFP利福布丁，PAS对氨基水杨酸，ETH乙硫异烟胺，CYC环丝氨酸，INH异烟肼，KAN卡那霉素，EMB乙胺丁醇；POS为阳性对照；浓度单位μg/mL。
从图2中可以看出，H37Rv△Rv1527c敲除菌对AMI(阿米卡星)、STR(链霉素)和KAN(卡那霉素)的敏感性增加，提示H37Rv△Rv1527c敲除菌对阿米卡星、链霉素、卡那霉素等氨基糖苷类抗结核药物具有较强的敏感性和特异性。
经过上述筛选，本实施例还针对属于氨基糖苷类抗结核药物的阿米卡星分别对H37Rv标准菌、H37Rv△Rv1527c敲除菌进一步进行最低抑菌浓度(MIC)试验。
配制好按倍比稀释的10个浓度(范围为0.12～64μg/mL)的阿米卡星药液并分别取100μL加入U底96孔板；分别制备0.5McFarland标准的H37Rv标准菌和H37Rv△Rv1527c敲除菌的菌悬液，分别取100μL菌悬液至11mL 7H9+10％OADC培养液涡旋振荡混匀30秒，再向96孔板中加入100μL 7H9+10％OADC菌悬液(H37Rv标准菌、H37Rv△Rv1527c敲除菌)，结果与实施例1中一致。证明H37Rv△Rv1527c敲除菌的突变基因的突变菌对属于氨基糖苷类抗结核药物的阿米卡星抗生素具有敏感性。
本实施例还分别取7H9+10％OADC含菌菌悬液(H37Rv标准菌和G1637A突变菌)各50μL在7H10+10％OADC平板上划线培养证明该培养物是单一微生物。通过菌落计数检验阳性对照孔中接种物浓度，菌落数为1×105CFU/mL，说明接种的菌浓度符合标准(标准范围5×104～5×105)。
实施例3
对结核分枝杆菌临床分离菌含有Rv1527c A1380P突变基因和Rv1527c G1637A突变基因的突变菌，按照实施例1的方法进行阿米卡星抗生素的最低抑菌浓度(MIC)试验。两种突变菌均表现出对阿米卡星抗生素具有较强的敏感性。
实施例4
本发明作为生物标志物的应用。
对一诊断MDR-TB的女性32岁患者，该患者痰标本分离出来的结核分枝杆菌含有Rv1527c G1637A突变基因，也就是说该临床分离菌Rv1527c基因片段上的第1637位的甘氨酸突变为丙氨酸。经二线抗结核药(方案中包括AMI)治疗，成功治愈。图3为患者二线抗结核药治疗前后胸部CT双肺病灶吸收情况，从图3中可以看出，A1～A2二线治疗前双肺多发斑片状影及多发空洞(箭头)；B1～B2治疗后2月，左肺空洞缩小，双肺斑片影较前吸收(箭头)；C1～C2治疗后8月，双肺斑片影仍较前吸收，右肺明显(箭头)；D1～D2治疗20月，疗程结束后7月，双肺斑片影及空洞较治疗前明显吸收，可见少部分结节、纤维条索状影及小空洞(箭头)。其中，A至D表示治疗前后4个不同时期患者双肺病变的情况，A1和A2表示同一时期双肺病变的不同层面，B1和B2、C1和C2以及D1和D2以此类推(注：每A1、A2、B1、B2等每一个图都是包括左和右的完整肺的不同层面和不同时期)。
由以上实施例可知，本发明提供了一种结核分枝杆菌的突变基因，并提供了该突变基因作为生物标志物的应用。本发明提供的三种突变基因均可提高结核分枝杆菌对AMI、STR、KAN等氨基糖苷类抗结核药的敏感性，可用临床治疗前的检测，指导临床用药，提高治愈率。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种结核分枝杆菌的突变基因，其特征在于，所述突变基因是在结核分枝杆菌标准株H37Rv基因中缺失了基因片段Rv1527c的突变基因，或结核分枝杆菌的Rv1527c基因片段上第1380位的丙氨酸突变为脯氨酸或第1637位的甘氨酸突变为丙氨酸的突变基因。
2.如权利要求1所述的一种结核分枝杆菌的突变基因，其特征在于，所述基因片段Rv1527c位于结核分枝杆菌标准株H37Rv基因中的第1722083～1728409位。
3.如权利要求1或2所述的一种结核分枝杆菌的突变基因，其特征在于，所述结核分枝杆菌标准株H37Rv的基因登录号为NC_000962.3。
4.权利要求1～3任意一项所述的突变基因作为结核分枝杆菌生物标志物的应用。
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