CN115969965A - 一种结核分枝杆菌的重组dna疫苗及其制备方法 - Google Patents
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Abstract
本发明公开了一种结核分枝杆菌的重组DNA疫苗及其制备方法。本发明的重组DNA疫苗通过将基因Ag85B、Rv2029c和Rv1738重组入真核表达载体获得。本发明研究发现，免疫6～8周龄C57BL/6雌性小鼠后，DNA疫苗Ag85B‑Rv2029c‑Rv1738‑pVAX1可以显著提高小鼠外周血中CD4+，CD8+T淋巴细胞的比例，增加小鼠脾淋巴细胞抗原特异性Th1型细胞因子IL‑2，IFN‑γ，TNF‑α的分泌，降低小鼠肺、脾的BCG细菌计数，提示该重组DNA疫苗可以有效控制分枝杆菌在小鼠体内的生长繁殖。因此，本发明的重组DNA疫苗可用于结核分枝杆菌感染的预防和治疗。
Description
本申请是申请日为2020年09月29日、申请号为CN202011047690.8、发明名称为《一种结核分枝杆菌的重组DNA疫苗及其制备方法》的分案申请。
技术领域
本发明涉及基因工程和重组DNA疫苗开发技术领域，具体涉及一种结核分枝杆菌的重组DNA疫苗及其制备方法。
背景技术
结核病(TB)是由结核分枝杆菌(MTB)引起的一种严重的呼吸道传染病。虽然多种类型的新型疫苗已被广泛研究，包括重组BCG疫苗、营养缺陷型结核分枝杆菌疫苗、蛋白质多肽疫苗、DNA疫苗、以病毒为载体的结核亚单位疫苗等，但现有疫苗或多或少存在缺陷，例如卡介苗(BCG)虽然可以有效预防粟粒型结核和结核性脑膜炎。但是其对成人保护效果有限，复种无效。且卡介苗为预防性疫苗，不能对潜伏感染的个体提供有效保护，因此，研制新型结核疫苗迫在眉睫。
发明内容
本发明的目的是提供一种结核分枝杆菌的重组DNA疫苗及其制备方法，该疫苗可补充市售卡介苗性能，控制结核病潜伏感染，有望用于卡介苗初始免疫后的加强免疫用疫苗；控制潜伏感染的治疗性疫苗；与药物联用控制结核病的治疗性疫苗。
本发明中，选取标准株H37Rv中完整的Ag85B，Rv2029c和Rv1738，通过酶切连接于表达载体；其中，重组的Rv1738(285bp,1965657..1965941)，Rv2029c(1020bp,2275405..2276424)可以用于潜伏期结核病的血清学检测，Ag85B(978bp，2134897..2135874)可以用于急性感染期的血清学检测，因此这些基因都可以作为疫苗的候选基因，本发明将上述基因重组于载体pVAX1，作为DNA疫苗。本发明的技术方案具体介绍如下。
一种结核分枝杆菌的重组DNA疫苗，其通过将基因Ag85B、Rv2029c和Rv1738重组入表达质粒获得。优选的，表达质粒为pVAX1，重组疫苗为Ag85B-Rv2029c-Rv1738-pVAX1(简称为B21)。
本发明进一步提供一种上述的结核分枝杆菌的重组DNA疫苗的制备方法，包括以下步骤：
(1)扩增Ag85B、Rv2029c和Rv1738基因；
(2)分别用双酶切Ag85B基因和表达质粒，胶回收连接获得重组质粒；
(3)将步骤(2)获得的重组质粒转化、扩增并表达；
(4)分别用双酶切步骤(3)获得的重组质粒和Rv2029c基因，胶回收连接形成重组质粒；
(5)将步骤(4)获得的重组质粒转化、扩增并表达；
(6)分别用双酶切步骤(5)获得的重组质粒和Rv1738基因，胶回收连接形成重组质粒；
(7)将步骤(6)获得的重组质粒转化、扩增并表达，即得到重组DNA疫苗。
优选的，步骤(2)中，表达质粒是pVAX1。
优选的，步骤(2)中，双酶切使用的酶是Nhe I和HindⅢ；步骤(4)中，双酶切使用的酶是HindⅢ和EcoRⅠ；步骤(6)中，双酶切使用的酶是EcoRⅠ和NotⅠ。
优选的，步骤(3)、步骤(5)和步骤(7)中，重组质粒在大肠杆菌中转化、扩增并表达。
和现有技术相比，本发明的有益效果在于：
本发明的疫苗中，使用美国FDA认可的核酸疫苗载体pVAX1，能够在哺乳动物细胞中表达蛋白。
DNA疫苗Ag85B-Rv2029c-Rv1738-pVAX1使用核酸疫苗载体pVAX1融合表达结核杆菌抗原Ag85B，Rv2029c和Rv1738，该疫苗转染巨噬细胞后，融合蛋白Ag85B-Rv2029c-Rv1738可以在巨噬细胞中表达，并有效激活巨噬细胞，提高IL-6，TNF-α的分泌水平。
本发明研究发现，免疫6-8周龄C57BL/6雌性小鼠2周后，该DNA疫苗Ag85B-Rv2029c-Rv1738-pVAX1可以显著提高小鼠外周血中CD4+，CD8+T淋巴细胞的比例，CD4+，CD8+T淋巴细胞在抗结核免疫过程中均有重要作用。
本发明研究发现，免疫6～8周龄C57BL/6雌性小鼠18周后，采用尾静脉注射5×106CFU BCG/只感染小鼠后4周，脾淋巴细胞在Ag85B，Rv2029c和Rv1738特异性抗原刺激后TH1型细胞因子IL-2，IFN-γ，TNF-α的分泌升高，而且疫苗免疫组中小鼠肺、脾的BCG计数显著降低，提示DNA疫苗可以有效控制结核分枝杆菌等在小鼠体内的生长繁殖，用于结核分枝杆菌感染的预防和治疗。
本发明研究用DNA疫苗Ag85B-Rv3425-Rv1813c-pVAX1(B31)作对照，发现该发明的DNA疫苗(B21)相比B31能更有效的激活巨噬细胞并提高TH1型细胞免疫反应。
附图说明
图1为酶切验证重组DNA疫苗的构建情况，其中，Lane1：DNAMarker，Lane2：对照载体pVAX1，Lane3：重组Ag85B-Rv2029c-Rv1738-pVAX1，Lane4：重组Ag85B-Rv3425-Rv1813-pVAX1；
图2为重组B21 DNA，B31 DNA疫苗在真核细胞中表达情况，其中，Lane1：蛋白Ag85B对照，Lane2：pVAX1载体转染，Lane3：B21转染，Lane：B31转染；
图3为重组B21 DNA疫苗转染Raw264.7细胞后的IL-6(a)，TNF-α(b)分泌结果；
图4为重组B21 DNA疫苗流式染色结果；
图5中(a)为采用BCG对小鼠进行攻毒后的细胞上清IL-2，IFN-γ，TNF-α含量检测结果；(b)为采用BCG对小鼠进行攻毒后的肺和脾CFU计数结果。
具体实施方式
本发明中未特定标注的试剂和条件均采用本领域普通技术人员熟知的常规产品和方法。
实施例1
重组DNA疫苗的制备
1.1结核分枝杆菌H37Rv菌株基因组DNA的制备
结核分枝杆菌(H37Rv)在7H9 Broth培养基中培养4周，80℃，2h灭活。用细菌DNA(小量)抽提试剂盒抽提基因组DNA。因结核菌的壁厚，细菌的消化时间延长至3到5h。
1.2结核分枝杆菌H37Rv菌株Ag85B，Rv2029c，Rv1738基因的扩增。
1)具体引物设计如下：
Ag85B-S(SEQ ID NO.1)：5’-AAAGCTAGCGCCACCATGGCATTCT CCCGGCCGGGGCT-3’，酶切位点为：NheⅠ
Ag85B-A(SEQ ID NO.2)：5’-TTTAAGCTTGCCGGCGCCTAACGAA CTCT-3’，酶切位点为：HindⅢ
Rv2029c-S(SEQ ID NO.3)：5’-TTTAAGCTTATGACGGAGCCAGCG GCGT-3’，酶切位点为：HindⅢ
Rv2029c-A(SEQ ID NO.4)：5’-AAAGAATTCTGGCGAGGCTTCCGG GTTAACGA-3’，酶切位点为：EcoRⅠ
Rv1738-S(SEQ ID NO.5)：5’-AAAGAATTCATGTGCGGCGACCAGT-3’，酶切位点为：EcoRⅠ
Rv1738-A(SEQ ID NO.6)：5’-AAAGCGGCCGCCTACTATCAATACA ACAATCGCGCCGG-3’，酶切位点为：NotⅠ
2)PCR反应体系(takaraPCR试剂)：
3)PCR反应条件
产物用1％的琼脂糖凝胶电泳鉴定。
1.3载体pVAX1、Ag85B的酶切、连接及转化
载体pVAX1、Ag85B的PCR产物使用NheⅠ和HindⅢ双酶切，37℃，3h。用胶回收试剂盒纯化酶切产物。T4 DNA连接酶4℃连接过夜。转化大肠杆菌E.coli DH5α菌株，挑取克隆抽质粒进行测序验证。
将上述构建成功载体、Rv2029c的PCR产物使用HindⅢ和EcoRⅠ双酶切，37℃，3h。用胶回收试剂盒纯化酶切产物。T4 DNA连接酶4℃连接过夜。转化大肠杆菌E.coli DH5α菌株，挑取克隆抽质粒进行测序验证。
将上述构建成功载体、Rv1738的PCR产物使用EcoRⅠ和NotⅠ双酶切，37℃，3h。用胶回收试剂盒纯化酶切产物。T4 DNA连接酶4℃连接过夜。转化大肠杆菌E.coli DH5α菌株，挑取克隆抽质粒进行测序验证。至此，重组的DNA疫苗Ag85B-Rv2029c-Rv1738-pVAX1(B21)构建完成(图1)。
同时使用上述方法，构建Ag85B-Rv3425-Rv1813c-pVAX1(B31)。
酶切验证重组DNA疫苗的构建情况如图1所示，Lane1：DNA Marker，Lane2：对照载体pVAX1，Lane3：重组Ag85B-Rv2029c-Rv1738-pVAX1，Lane4：重组Ag85B-Rv3425-Rv1813c-pVAX1。使用限制性内切酶NheⅠ和NotⅠ酶切。从图中可以看出，重组质粒B21携带有2163bp外源片段，重组质粒B31携带有1823bp的片段。表明重组质粒重组Ag85B-Rv2029c-Rv1738-pVAX1，Ag85B-Rv3425-Rv1813c-pVAX1构建成功。
1.4重组DNA疫苗细胞表达的验证
用上述构建成功的DNA疫苗质粒转染HEK293t细胞，收集细胞样品，检测是否真核表达。具体为，将12孔板中每孔种满1×106HEK293t细胞过夜，在无血清培养基条件下每孔使用3μL Lipofectamine 2000(Thermo Scientific)转染2μL质粒，4h后换用DMEM+10％FBS培养48h收取细胞样本，分别使用anti-Ag85B抗体用于westernblot检测。
如图2所示，Lane1：蛋白Ag85B对照，Lane2：pVAX1载体转染，Lane3：B21转染，Lane：B31转染。结果表明：转染48h后，重组B31 DNA疫苗在真核细胞中表达融合蛋白Ag85B-Rv3425-Rv1813c，重组B21 DNA疫苗在真核细胞中表达融合蛋白Ag85B-Rv2029c-Rv1738，而空载组在对应位置均没有条带。
实施例2
疫苗细胞水平免疫及免疫指标的测定
DNA疫苗质粒转染Raw264.7细胞，收集培养上清、细胞样品，检测相关免疫指标。具体为，将24孔板中每孔种满2×105Raw264.7细胞过夜，在无血清培养基条件下每孔使用1μLjet OPTIMUS(polypuls)转染1μg质粒，4h后换用DMEM+10％FBS培养24h、48h、72h收取细胞上清。
取处理后的细胞上清，使用Mouse IL-6ELISA Kit，Mouse TNF-αELISA Kit(达科为)按照试剂盒内说明进行检测。如图3所示，经过比较发现，进行转染后，本发明的DNA疫苗B21相比DNA疫苗B31可以更有效的诱导巨噬细胞分泌IL-6，TNF-α，而且相对于PBS对照组IL-6，TNF-α的分泌量增加显著。
实施例3
疫苗动物水平免疫指标及保护水平
将重组疫苗免疫6-8周龄C57BL/6雌性小鼠后，每两周免疫一次，共免疫三次。免疫结束后两周，眼眶取血，使用红细胞裂解液裂红后，检测外周血中CD4+，CD8+T淋巴细胞的比例，如图4所示，流式染色结果表明：重组DNA疫苗B21可以促进小鼠外周血中CD4+和CD8+T细胞比例上升，相对于PBS对照组差异显著。
免疫结束后18周，采用尾静脉注射BCG 5×106CFU/只，4周后解剖。分离脾淋巴细胞，分别用5μg/ml Ag85B，Rv2029c，Rv1738蛋白刺激淋巴细胞，36h后检测细胞上清中IL-2，IFN-γ，TNF-α的分泌水平(图5a)。结果显示，DNA疫苗B21组细胞因子分泌水平相较于PBS组显著上升。同时进行了肺、脾CFU计数(图5b)，相比PBS组，DNA疫苗B21组显著降低了肺、脾的载菌量。且图5显示DNA疫苗B21组脾淋巴细胞分泌细胞因子高于B31组，肺、脾的载菌量低于B31组。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (8)
1.Ag85B基因、Rv2029c基因和Rv1738基因在制备治疗结核病的重组DNA疫苗中的应用。
2.Ag85B基因、Rv2029c基因和Rv1738基因在制备诱导巨噬细胞分泌IL-6和TNF-α的重组DNA疫苗中的应用。
3.Ag85B基因、Rv2029c基因和Rv1738基因在制备诱导淋巴细胞分泌IL-2，IFN-γ和TNF-α的重组DNA疫苗中的应用。
4.根据权利要求1～3任一项所述的应用，其特征在于，所述重组DNA疫苗通过将Ag85B基因、Rv2029c基因和Rv1738基因重组入真核表达载体获得。
5.根据权利要求4所述的应用，其特征在于，所述真核表达载体为pVAX1，重组DNA疫苗为Ag85B-Rv2029c-Rv1738-pVAX1。
6.根据权利要求4所述的应用，其特征在于，所述重组DNA疫苗的制备方法，包括以下步骤：
(1)扩增Ag85B、Rv2029c和Rv1738基因；
(2)分别用双酶切Ag85B基因和表达质粒，胶回收连接获得重组质粒；
(3)将步骤(2)获得的重组质粒转化、扩增并表达；
(4)分别用双酶切步骤(3)获得的重组质粒和Rv2029c基因，胶回收连接形成重组质粒；
(5)将步骤(4)获得的重组质粒转化、扩增并表达；
(6)分别用双酶切步骤(5)获得的重组质粒和Rv1738基因，胶回收连接形成重组质粒；
(7)将步骤(6)获得的重组质粒转化、扩增并表达，得到重组DNA疫苗。
7.根据权利要求6所述的应用，其特征在于，步骤(2)中，双酶切使用的酶是NheⅠ和HindⅢ；步骤(4)中，双酶切使用的酶是HindⅢ和EcoRⅠ；步骤(6)中，双酶切使用的酶是EcoRⅠ和NotⅠ。
8.根据权利要求6所述的应用，其特征在于，步骤(3)、步骤(5)和步骤(7)中，重组质粒在大肠杆菌中转化、扩增并表达。
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