CN113943689B - 一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用 - Google Patents
一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用 Download PDFInfo
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Abstract
本发明公开了一种免疫激活性重组乳酸乳球菌的构建及作为肿瘤疫苗和免疫佐剂等方面的应用，属于生物医学及生物工程领域。两种重组乳酸乳球菌均可在菌体相应位置表达Flt3L‑OX40L融合蛋白。成功构建后经反复实验验证，两种重组乳酸乳球菌疫苗不仅可以有效活化树突状细胞(DC)，而且可以促进T细胞分化成免疫记忆性效应细胞(TEM)。动物实验提示原位瘤内注射任意一种本发明的免疫激活性重组乳酸乳球菌具有抑制肿瘤乃至使肿瘤完全消失的效果。
Description
技术领域
本申请涉及生物医学及生物工程领域，具体而言，涉及一种胞内表达或胞壁锚定表达 Flt3L-OX40L融合蛋白的重组乳酸乳球菌的构建方法和应用。
背景技术
肿瘤免疫治疗是迄今肿瘤治疗中最受瞩目、最具临床潜力的抗肿瘤治疗领域。近10年来，肿瘤免疫治疗主要在以下四方面取得卓越成就：(1)免疫检查点调控因子如PD1抑制剂等；(2)工程化T细胞的过继免疫治疗如TCR-T、CAR-T等；(3)抗体-药物偶联物(Antibody-drugconjugates,ADC)如由单耳他汀E(MMAE)与抗CD30人鼠嵌合抗体Brentuximab 偶联而形成的药物等；(4)肿瘤疫苗如新抗原疫苗、原位疫苗等。尽管如此，实体肿瘤的免疫治疗仍有许多挑战，如肿瘤微环境中免疫活性细胞过少，抑制性因素过多；新抗原异质性太大，难以找到优质新抗原靶点等。因此，原位瘤内注射即原位疫苗可以充分改善肿瘤微环境，使肿瘤自身成为“疫苗工厂”，进而能够源源不断地激活抗肿瘤免疫反应。
合成生物学是一门涉及生物化学、分子生物学、基因工程及计算机科学等多个领域的新型综合性交叉学科。其目的是通过合成生物功能元件、装置、系统，对生命体进行有目标的遗传学设计、改造，使细胞和生物体产生特定生物功能，乃至合成“人造生命”。其在肿瘤免疫治疗发展前景主要包括工程化细胞、工程化病毒、工程化细菌等。其中，细菌因为具有易培养、易扩增、易改造等优点而受到研究者们的青睐。改造后的细菌可具有靶向性、有效性、安全性。目前细菌的主要选择包括减毒沙门氏菌、大肠杆菌、芽孢杆菌、单核增生性李斯特菌、乳酸菌等等。乳酸菌作为益生菌，相对而言更加安全。因此，乳酸菌或许是合成生物学改造的较佳对象。
肿瘤治疗的失败往往由于肿瘤微环境的免疫抑制因素过多，且免疫活性细胞如DC和T 细胞在肿瘤局部过少或者无法充分发挥特异性免疫细胞毒性效应。故合理地瘤内注射免疫佐剂可以改善上述缺点。Flt3L作为最主要的造血生长因子之一，能在体内外明显地扩增DC、 NK细胞和T淋巴细胞的数量。OX40(CD134)是TNFR超家族成员之一，可激活经典的NF-κB1 途径或非经典的NF-κB2途径、PI3k/PKB和NFAT途径，对于细胞存活至关重要。当OX40与其配体OX40L结合时，可促进效应T细胞和记忆T细胞的存活和扩增，增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌，降低Tregs的免疫抑制活性。乳酸乳球菌可以激活机体固有免疫功能。因此，胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌疫苗具有改善肿瘤微环境，进而达到抗肿瘤免疫治疗的潜力。
发明内容
本发明的目的是充分调动肿瘤微环境中的免疫正性因素，改善免疫负性调节，因此构建了一种胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌疫苗，同时提供该疫苗的改造方法及其作为抗肿瘤药物的应用。
本发明公开了一种免疫激活性重组乳酸乳球菌，该免疫激活性重组乳酸乳球菌为胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌疫苗，所述融合蛋白包括Flt3L和 OX40L，锚定所用蛋白为N-乙酰胞壁质酶AcmA的C端重复序列，蛋白之间经由连接肽 PVGLIG连接组成，胞内表达的融合方式为Flt3L-OX40L，胞壁锚定的融合方式为Flt3L-OX40L-cAcmA。
Flt3L-OX40L融合蛋白中，融合蛋白为Flt3L胞外域或其他能结合Flt3的分子，OX40L 为OX40L、OX40单克隆抗体或其他能活化OX40的分子，AcmA为AcmA的C端重复序列或其他能与乳酸乳球菌细胞壁锚定结合的蛋白分子。
Flt3L-OX40L融合蛋白中，所述融合蛋白具有如下氨基酸序列：由SEQ ID No.1所示的氨基酸序列组成的蛋白质。
Flt3L-OX40L融合蛋白中，所述融合蛋白具有如下核苷酸序列：由SEQ ID No.2所示的核苷酸序列。
Flt3L-OX40L-cAcmA融合蛋白中，所述融合蛋白具有如下氨基酸序列：由SEQ IDNo.3 所示的氨基酸序列组成的蛋白质。
Flt3L-OX40L-cAcmA融合蛋白中，所述融合蛋白具有如下核苷酸序列：由SEQ IDNo.4 所示的核苷酸序列。
本发明还提供了上述免疫激活性重组乳酸乳球菌作为肿瘤疫苗或免疫佐剂的应用。
进一步的，所述肿瘤为结肠癌、胃癌、乳腺癌、肝癌、肺癌或宫颈癌肿瘤。
本发明还提供了上述免疫激活性重组乳酸乳球菌的构建方法，包括以下步骤：
将SEQ ID NO.2和SEQ ID NO.4所示的基因片段通过酶切、连接、同源重组，克隆到pNZ8148载体上，得到pNZ8148-Flt3L-OX40L和pNZ8148-Flt3L-OX40L-cAcmA；
将所述pNZ8148-Flt3L-OX40L或pNZ8148-Flt3L-OX40L-cAcmA重组载体转化进表达宿主细胞，通过培养和诱导剂诱导表达，得到胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌。
进一步的，所述表达宿主细胞为乳酸乳球菌NZ9000菌株；
作为优选的方案，所述诱导剂为Nisin诱导剂，所述Nisin诱导剂的终浓度为10ng/ml。
本发明的有益效果是：
Flt3L能在体内外明显地扩增DC、NK细胞和T淋巴细胞的数量。OX40(CD134)是TNFR超家族成员之一，当OX40与其配体OX40L结合时，可促进效应T细胞和记忆T细胞的存活和扩增，增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌，降低Tregs的免疫抑制活性。乳酸乳球菌可以激活机体固有免疫功能。因此，原位瘤内注射该工程化细菌具有改善肿瘤微环境，进而达到抗肿瘤免疫治疗的潜力。
附图说明
图1是表达载体pNZ8148-Flt3L-OX40L中NcoⅠ和HindⅢ位点之间序列的测序结果示意图。图2是表达载体pNZ8148-Flt3L-OX40L-cAcmA中NcoⅠ和HindⅢ位点之间序列的测序结果示意图。
图3是重组乳酸乳球菌pNZ8148-Flt3L-OX40L的诱导表达结果图。
图4是重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA的诱导表达结果图。
图5是流式检测重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA的锚定效率的结果图。
图6是体外验证小鼠骨髓来源细胞BMDC活化效率的流式检测结果图。
图7是体外验证小鼠骨髓来源细胞BMDC活化效率的流式检测结果统计图。
图8是体外验证小鼠脾脏细胞TEM活化效率的流式检测结果图。
图9是体外验证小鼠脾脏细胞TEM活化效率的流式检测结果统计图。
图10是体内验证肿瘤内原位注射重组乳酸乳球菌的抑瘤效果图。
具体实施方式
以下结合实施例及附图进一步详细描述本发明。然而应当理解，这些实施例只是为起说明作用，而非用于限制本发明。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
乳酸乳球菌pNZ8148-Flt3L-OX40L的构建：
根据SEQ ID NO.1，设计优化其编码基因的基因序列如SEQ ID NO.2所示，并设计相应的酶切位点。
提取pNZ8148载体质粒，并用NcoI/HindIII双酶切pNZ8148载体质粒和目的基因；37℃， 2h；然后用1％琼脂糖凝胶电泳，观察电泳结果。利用DNA回收试剂盒回收目的基因片段和 pNZ8148载体质粒双酶切产物。将回收的pNZ8148载体质粒线性化片段和目的基因片段通过互补的粘性末端，在T4DNA连接酶的作用下连接成闭合环状的DNA分子，即形成重组质粒pNZ8148-Flt3L-OX40L。
将连接完成的重组质粒送样进行测序，测序结果准确，且无移码突变，结果如图1所示。
实施例2
乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA的构建：
根据SEQ ID NO.3，设计优化其编码基因的基因序列如SEQ ID NO.4所示，并设计相应的酶切位点。
提取pNZ8148载体质粒，并用NcoI/HindIII双酶切pNZ8148载体质粒和目的基因；37℃， 2h；然后用1％琼脂糖凝胶电泳，观察电泳结果。利用DNA回收试剂盒回收目的基因片段和 pNZ8148载体质粒双酶切产物。将回收的pNZ8148载体质粒线性化片段和目的基因片段通过互补的粘性末端，在T4DNA连接酶的作用下连接成闭合环状的DNA分子，即形成重组质粒pNZ8148-Flt3L-OX40L-cAcmA。
将连接完成的重组质粒送样进行测序，测序结果准确，且无移码突变，结果如图2所示。
实施例3
重组乳酸乳球菌pNZ8148-Flt3L-OX40L的表达和检测：
将重组质粒pNZ8148-Flt3L-OX40L转化乳酸菌进行蛋白表达和检测。
(1)pNZ8148-Flt3L-OX40L的诱导表达：将验证正确的重组表达质粒 pNZ8148-Flt3L-OX40L电转化进乳酸球菌NZ9000中，得到重组乳酸菌pNZ8148-Flt3L-OX40L，将阳性克隆子(pNZ8148-Flt3L-OX40L)接种于 5mLM17+0.5％glucose+10μg/mL chloramphenicol培养液的试管中，30℃过夜静置培养；次日按1:100接种于M17+0.5％glucose+10μg/mL的chloramphenicol培养液中，30℃静置培养至菌体OD600约为0.3～0.5；加入Nisin至终浓度为10ng/mL，30℃培养3～4h，诱导融合蛋白表达。
(2)Western blot检测pNZ8148-Flt3L-OX40L的表达情况：pNZ8148-Flt3L-OX40L经诱导表达后，取出1mL培养物，12000g/min室温离心5min，沉淀用200μL 1×上样缓冲液重悬，煮沸5分钟后Western Blot检测分析，结果如图3所示。
实施例4
重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA的表达和检测：
将重组质粒pNZ8148-Flt3L-OX40L-cAcmA转化乳酸菌进行蛋白表达和检测。
(1)pNZ8148-Flt3L-OX40L-cAcmA的诱导表达：将验证正确的重组表达质粒pNZ8148-Flt3L-OX40L-cAcmA电转化进乳酸球菌NZ9000中，得到重组乳酸菌 pNZ8148-Flt3L-OX40L-cAcmA，将阳性克隆子(pNZ8148-Flt3L-OX40L-cAcmA)接种于 5mLM17+0.5％glucose+10μg/mL chloramphenicol培养液的试管中，30℃过夜静置培养；次日按1:100接种于M17+0.5％glucose+10μg/mL的chloramphenicol培养液中，30℃静置培养至菌体OD600约为0.3～0.5；加入Nisin至终浓度为10ng/mL，30℃培养3～4h，诱导融合蛋白表达。
(2)Western blot检测pNZ8148-Flt3L-OX40L-cAcmA的表达情况：pNZ8148-Flt3L-OX40L 经诱导表达后，取出1mL培养物，12000g/min室温离心5min，沉淀用200μL 1×上样缓冲液重悬，煮沸5分钟后Western Blot检测分析，结果如图4所示。
实施例5
重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA的抗原表面展示性能的鉴定：
(1)重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA诱导表达后取20ul菌液，野生乳酸乳球菌同样培养条件下取20ul菌液，1ml 2％BSA封闭液分别重悬上述两种菌，12000g/min 5min 离心，弃上清，重复洗涤一次。
(2)向两管细菌分别加100ul HIS-488抗体稀释液，避光孵育30分钟，后1ml 2％BSA封闭液分别重悬上述两种菌，12000g/min 5min离心，弃上清，重复洗涤一次。
(3)流式检测HIS-488荧光，结果如图5所示，提示重组乳酸乳球菌 pNZ8148-Flt3L-OX40L-cAcmA可成功表达融合蛋白Flt3L-OX40L并将其锚定在细菌壁表面。
实施例6
体外验证小鼠骨髓来源细胞BMDC活化效率：
(1)BALB/c小鼠脱颈处死后，泡入酒精，手术取出所有股骨和胫骨，剪刀和镊子将骨周围的肌肉组织尽量去除干净。将骨移至超净台内，并用盛有70％酒精的无菌培养皿浸泡2-5 分钟，以消毒灭菌，然后用无菌的NS洗2次。将骨移入另一个盛有NS的新培养皿中，用剪刀减去骨两端，再用注射器抽取NS，针头分别从骨两端插入骨髓腔，反复冲洗骨髓至培养皿中，直至骨完全变白。收集骨髓悬液，用40um的滤器过滤，除去小碎片和肌肉组织。滤过液1200rpm离心5min，弃上清。加入2ml红细胞裂解液(约3倍体积)，重悬细胞，室温孵育3-5min，最长10min。加入10ml NS中和裂解液的作用，然后1200rpm离心5min，弃上清。NS洗一次，然后用含10％FBS的RPMI 1640培养液重悬细胞，至此已获得小鼠骨髓细胞。
(2)将上述DC用1640+10％FBS调整至5*10^5cell/ml。第0天，铺至24孔板，每孔1ml细胞，同时加入rm GM-CSF 20ng/ml，和IL-4 10ng/ml，孵箱培养，胶布封防止液体流失。
(3)第2天和第4天，3/4换液，补足rm GM-CSF和rm IL-4。
(4)第6天，轻柔吹打培养液，收集悬浮细胞及疏松贴壁生长的细胞。1200rpm离心5min，弃上清。用含1640+10％FBS重悬细胞并计数，然后调整至1*10^6cell/ml。并加入rmGM-CSF (20ng/ml)和IL-4(10ng/ml)。铺至6孔板。
(5)第8天，收集悬浮细胞，以1200rpm，5min离心，弃上清。用1640+10％FBS重悬细胞至1*10^6cell/ml，铺至24孔板。设计5组，每组铺3孔。分别为阴参生理盐水NS组、阳参LPS(1ug/ml)、野生菌(10^7CFU每孔)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L(10^7 CFU每孔)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA(10^7CFU每孔)。孵箱培养，胶布封防止液体流失。
(6)第10天，分别收集每孔细胞于流式管中。每管中加2ul CD11c-FITC抗体，1ulCD80-APC抗体、CD86-PE抗体。避光孵育30分钟，分别每管加1ml NS，1200rpm，5min 离心，弃上清。重复洗涤一次。流式检测分析结果。
(7)结果如图6、7所示，重组乳酸乳球菌pNZ8148-Flt3L-OX40L(10^7CFU每孔)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA(10^7CFU每孔)均可显著刺激DC细胞的活化。
实施例7
体外验证小鼠脾脏细胞TEM活化效率：
(1)第0天，BALB/c小鼠脱颈处死后，泡入酒精，手术取出脾脏，在超净台内物理研磨，收集脾脏悬液，用40um的滤器过滤，除去筋膜组织。滤过液1200rpm离心5min，弃上清。加入2ml红细胞裂解液(约3倍体积)，重悬细胞，室温孵育3-5min，最长10min。加入10ml NS中和裂解液的作用，然后1200rpm离心5min，弃上清。NS洗一次，然后用含0.5％血清的AIMV(内含40ng/ml的GM-CSF和20ng/ml的IL-4)培养液重悬细胞，计数，配成浓度6*10^6/ml。取96孔U型板，每孔加入100ul 6*10^6/ml的细胞。
(2)第1天，每3孔为一组。A组加NS 10ul；B组加6*10^5个CT26肿瘤细胞反复冻融后裂解物及10^7CFU野生乳酸乳球菌NZ9000；C组加6*10^5个CT26肿瘤细胞反复冻融后裂解物及10^7CFU重组乳酸乳球菌pNZ8148-Flt3L-OX40L；D组加6*10^5个CT26肿瘤细胞反复冻融后裂解物及10^7CFU重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA。4h后，分别每组加3ug/ml的R848。30min后，分别每组加50ng/ml的LPS。
(3)第2天，96孔板离心后去除全部上清，PBS清洗。用含50ng/ml IL-7和24IU/mlIL-2 的AIMV重悬至5*10^5/ml。铺至新96孔板，每孔200ul，即每孔含1*10^5细胞。
(4)第3天至第12天，用含50ng/ml IL-7和24IU/ml IL-2的AIMV半量换液。2-3天换一次液。根据细胞状态可做换液频率调整。
(5)BALB/c小鼠脱颈处死后，泡入酒精，手术取出脾脏，重复第0天至第2天的操作。
(6)原脾脏细胞中T细胞增殖，原浓度5*10^5/ml可达1*10^6/ml以上，200微升体系可含细2*10^5以上，全量换液并调整浓度至2*10^6/ml，以每孔100ul加入新96孔板。
(7)将(5)中刺激后的脾脏细胞全量换液并配平至1*10^6/ml，以每孔100ul加入上述 (6)中新96孔板。共孵育18～24h。
(8)分别收集每孔细胞于流式管中。每管中加2ul CD3-FITC抗体，1ul CD8-APC抗体、 CD44-PE抗体、CD62L-PE/Cy7抗体。避光孵育30分钟，分别每管加1ml NS，1200rpm，5min离心，弃上清。重复洗涤一次。流式检测分析结果。
(9)结果如图8、9所示，重组乳酸乳球菌pNZ8148-Flt3L-OX40L(10^7CFU每孔)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA(10^7CFU每孔)均可显著刺激TEM细胞的活化。
实施例8
体内验证肿瘤内原位注射重组乳酸乳球菌的抑瘤效果：
(1)小鼠实验流程：实验分组：PBS对照组(A)、野生菌NZ9000组(B)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L组(C)、重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA组(D)。每组8只小鼠，共32只。
(2)小鼠肿瘤模型：CT26结肠癌细胞重悬至5*10^6/ml，后每只小鼠皮下接种100ulCT26，即5*10^5个细胞，约一周肿瘤大小可生长至80～100mm^3。一周后开始治疗。
(3)小鼠免疫：经Nisin诱导培养后的乳酸菌，12000g/min，离心5min，弃上清收集菌体，用PBS缓冲溶液将沉淀洗涤两次，最后重悬调整浓度至1×10^9CFU/mL备用。分别于1，3，5天原位瘤内注射各组乳酸菌，共计3次，每次100μL/只，浓度为1×10^9CFU/mL (即1×10^8CFU)。
(4)每周用游标卡尺记录3～4次肿瘤大小，肿瘤体积为长*宽*宽/2。当小鼠肿瘤体积大于1500mm^3时，对小鼠实施安乐死。用体重秤记录小鼠体重评估健康状况。
(5)抑瘤及体重情况如图10所示，提示原位瘤内注射重组乳酸乳球菌 pNZ8148-Flt3L-OX40L组、重组乳酸乳球菌pNZ8148-Flt3L-OX40L-cAcmA组，可显著抑制肿瘤生长乃至使肿瘤完全消失。
以上所述实例是本发明申请的一部分实例，而非全部实施例。任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，依据本发明的技术实质，对以上实施例所作的任何简单的修改、等同替换与改进等，均仍属于本发明技术方案的保护范围之内。
序列表
<110> 南京鼓楼医院
<120> 一种基于重组乳酸乳球菌的肿瘤疫苗的构建方法及其应用
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Claims (7)
1.一种免疫激活性重组乳酸乳球菌, 其特征在于：该免疫激活性重组乳酸乳球菌为胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌疫苗，所述融合蛋白包括Flt3L和OX40L，锚定所用蛋白为N-乙酰胞壁质酶AcmA的C端重复序列，蛋白之间经由连接肽PVGLIG连接组成；胞内表达的融合方式为Flt3L-OX40L，胞壁锚定的融合方式为Flt3L-OX40L-cAcmA；Flt3L-OX40L融合蛋白中，所述融合蛋白由如下核苷酸序列编码：由SEQ IDNo.2所示的核苷酸序列；Flt3L-OX40L-cAcmA融合蛋白中，所述融合蛋白由如下核苷酸序列编码：由SEQ ID No.4所示的核苷酸序列。
2.根据权利要求1所述的免疫激活性重组乳酸乳球菌，其特征在于：Flt3L-OX40L融合蛋白中，所述融合蛋白具有如下氨基酸序列：由SEQ ID No.1所示的氨基酸序列组成的蛋白质。
3.根据权利要求1所述的免疫激活性重组乳酸乳球菌，其特征在于：Flt3L-OX40L-cAcmA融合蛋白中，所述融合蛋白具有如下氨基酸序列：由SEQ ID No.3所示的氨基酸序列组成的蛋白质。
4.权利要求1-3任一项所述的免疫激活性重组乳酸乳球菌在制备肿瘤疫苗中的应用。
5.根据权利要求4所述的免疫激活性重组乳酸乳球菌在制备肿瘤疫苗中的应用，其特征在于：所述肿瘤为结肠癌、胃癌、乳腺癌、肝癌、肺癌或宫颈癌肿瘤。
6.权利要求1-3任一项的免疫激活性重组乳酸乳球菌的构建方法，其特征在于，包括以下步骤：
将SEQ ID NO.2和SEQ ID NO.4所示的基因片段通过酶切、连接、同源重组，克隆到pNZ8148载体上，得到pNZ8148-Flt3L-OX40L和pNZ8148-Flt3L-OX40L-cAcmA；
将所述pNZ8148-Flt3L-OX40L和pNZ8148-Flt3L-OX40L-cAcmA重组载体转化进表达宿主细胞，通过培养和诱导剂诱导表达，得到胞内表达或胞壁锚定表达Flt3L-OX40L融合蛋白的重组乳酸乳球菌。
7.根据权利要求6所述的免疫激活性重组乳酸乳球菌的构建方法，其特征在于，所述表达宿主细胞为乳酸乳球菌NZ9000菌株；所述诱导剂为Nisin诱导剂，所述Nisin诱导剂的终浓度为10ng/ml。
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