CN116574685B - 一种装载抗菌肽ll-37的外泌体及其在抗寨卡病毒中的应用 - Google Patents
一种装载抗菌肽ll-37的外泌体及其在抗寨卡病毒中的应用 Download PDFInfo
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Abstract
本发明公开了一种装载抗菌肽LL‑37的外泌体及其在抗寨卡病毒中的应用,其中,抗菌肽LL‑37通过修饰在其N端或C端的跨膜蛋白装载于外泌体膜上,制备得到的产品安全性好,血清稳定性也大大提高,无论在体内还是体外都有显著的抗病毒作用,尤其是LL‑37‑TM‑exo,在对ZIKV感染的预防和治疗上均有极为优异的效果,可以作为防治寨卡病毒感染药物的有力备选。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种装载抗菌肽LL-37的外泌体及其在抗寨卡病毒中的应用。
背景技术
寨卡病毒属于黄病毒科,黄病毒属,1947年科学家们在乌干达首都附近的树林地区首次分离发现寨卡病毒。寨卡病毒的基因组为长度约10.1kb的单股正链RNA,可编码三个结构蛋白(C、prM/M和E)和七个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。病毒核酸被膜蛋白包裹形成直径40-50nm的病毒颗粒,呈二十面体结构。寨卡病毒主要是通过蚊虫叮咬传播,体内含有寨卡病毒的埃及伊蚊为主要传播媒介。同时研究证实,被寨卡病毒感染的患者也可经过其它方式传播给他人,如通过性接触和输血传播,还可以通过垂直传播由孕妇传给胎儿。根据医务工作者研究并经世界卫生组织证实,妊娠早期感染寨卡病毒可能导致新生儿小头畸形的发生;同时寨卡病毒可以引发大龄儿童及成年人出现格林巴利综合征、脊髓炎等比较严重的神经受损症状。然而目前尚无有效的疫苗或治疗药物来预防或治疗寨卡感染。
抗菌肽最初是由科学家在天蚕蛹中发现的一类阳离子小肽类物质,因为其具有杀灭病原菌的作用,便被命名为抗菌肽。抗菌肽不仅具有广谱的抗微生物活性,还具有许多其他生物学功能,如调节炎症反应、调节免疫、调控凋亡、杀灭肿瘤细胞以及参与组织修复中的信号通路等。大部分抗菌肽都带正电荷,具有两亲性,且分子量较小。
LL-37是人源cathelicidin家族抗菌肽Hcap-18前体蛋白上C末端剪切下来的成熟肽,氨基酸序列为LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPR TES,由37个氨基酸组成。现有的研究显示,LL-37具有(1)直接杀死病原菌,包括细菌和真菌(2)具有免疫调节作用(3)刺激血管生成,促进损伤修复(4)诱导细胞凋亡(5)细胞趋化作用等。此外,LL-37还具有广谱的抗病毒活性,包括人类获得性免疫缺陷病毒(human immune deficiency virus,HIV-1)、人腺病毒(human adenovirus,HAdV)、甲型流感病毒(influenza avirus,IAV)、呼吸道合胞病毒(respiratory syncytialvirus,RSV)、鼻病毒(rhinovirus,HRV)、牛痘病毒(vacciniavirus,VACV)、单纯疱疹病毒(herpesvirus hominis,HSV)和丙型肝炎病毒(hepatitis c virus,HCV)等,主要通过与细胞膜和病毒外壳之间的相互作用、增加I型干扰素表达、减少促炎细胞因子的产生而发挥抗病毒作用。随着科研人员对于抗菌肽的继续研究,抗菌肽的功能有待被更进一步的挖掘出来,为人类预防及治疗疾病发挥更大的作用。
发明内容
为解决上述问题,本发明提供的装载LL-37的外泌体血清稳定性大大提高,在体内体外都有显著的抗病毒作用,在防治寨卡病毒感染的药物研发中具有较好的应用前景,解决了目前尚无有效的疫苗和抗病毒药物可用于临床上寨卡病毒感染防治的问题。
本发明的第一个目的是提供一种装载抗菌肽LL-37的外泌体,所述抗菌肽LL-37通过修饰在其N端或C端的跨膜蛋白装载于外泌体膜上。
进一步地,所述跨膜蛋白的氨基酸序列如SEQ ID NO.1所示。
进一步地,所述跨膜蛋白的核苷酸序列如SEQ ID NO.2所示。
进一步地,所述抗菌肽LL-37的氨基酸序列如SEQ ID NO.3所示。
进一步地,所述抗菌肽LL-37的核苷酸序列如SEQ ID NO.4所示。
进一步地,所述装载抗菌肽LL-37的外泌体由以下步骤制备得到:
将跨膜蛋白编码基因融合至抗菌肽LL-37编码基因的5’端或3’端,再与表达载体连接得到重组质粒,将重组质粒转入宿主细胞中,构建得到重组细胞,并将该重组细胞制备成外泌体。
进一步地,通过差速离心法、TFF切向流过滤法、密度梯度离心、空间排阻色谱法、聚合物沉淀或免疫亲和等方法制备得到装载抗菌肽LL-37的外泌体。
本发明的第二个目的是提供上述装载抗菌肽LL-37的外泌体在制备抗寨卡病毒制剂中的应用。
进一步地,所述抗寨卡病毒制剂用于预防或治疗寨卡病毒感染。
本发明的第三个目的是提供一种抗寨卡病毒的药物,包括上述装载抗菌肽LL-37的外泌体。
本发明的有益效果:
本发明通过将抗菌肽LL-37以跨膜肽的方式装载于外泌体上,制备得到的产品安全性好,血清稳定性也大大提高,无论在体内还是体外都有显著的抗病毒作用,尤其是LL-37-TM-exo,在对ZIKV感染的预防和治疗上均有极为优异的效果,可以作为防治寨卡病毒感染药物的有力备选。
附图说明
图1为TM序列跨膜区域分析;
图2为本发明的装载LL-37外泌体的示意图;
图3为质粒设计及验证质粒过表达;其中,(A):转染后细胞中的荧光情况证明质粒过表达;(B):提取转染后细胞的全细胞裂解物,Western Blotting验证质粒过表达;
图4为外泌体中成功表达LL-37;其中,(A):提取转染后的细胞的膜蛋白,证明转染后LL-37定位在细胞膜中;(B):外泌体的Western Blotting结果表明LL-37在外泌体中表达;
图5为外泌体的NTA分析;
图6为LL-37-TM-exo的定量实验;其中,(A):100μg的LL-37-TM-exo中大约含有2.5μg的LL-37(Western Blotting图);(B):100μg的LL-37-TM-exo中大约含有2.5μg的LL-37(灰度分析图);
图7为装载LL-37的外泌体对细胞活力的影响;其中,(A):vector-exo、TM-LL-37-exo、LL-37-TM-exo对Vero E6细胞活力的影响;(B):vector-exo、LL-37-TM-exo对A549细胞活力的影响;(C):vector-exo、LL-37-TM-exo对HeLa细胞活力的影响;(D):vector-exo、LL-37-TM-exo对293T细胞活力的影响;(E):vector-exo、LL-37-TM-exo对U251细胞活力的影响;
图8为实时荧光定量PCR检测TM-LL-37-exo、LL-37-TM-exo对病毒RNA合成的影响;
图9为Western Blotting检测TM-LL-37-exo、LL-37-TM-exo对病毒蛋白NS3的表达的影响;其中,(A)TM-LL-37-exo、LL-37-TM-exo在体外能抑制病毒蛋白NS3的表达(WesternBlotting图);(B)TM-LL-37-exo、LL-37-TM-exo在体外能抑制病毒蛋白NS3的表达(灰度分析图);
图10为C57BL/6J雄鼠各个组织中的病毒载量;
图11为LL-37-TM-exo和LL-37的血清稳定性分析;其中,(A&B):LL-37和LL-37-TM-exo与血清共孵育1h时,对病毒NS3蛋白表达水平的影响;(C&D):LL-37和LL-37-TM-exo与血清共孵育6h时,对病毒NS3蛋白表达水平的影响;
图12为C57BL/6J小鼠各个组织中的病毒载量;
图13为C57BL/6J小鼠各个组织中的病毒载量;
图14为LL-37-TM-exo与LL-37的抗病毒效果比较;
图15为Western Blotting对病毒进行复制动力学检测的具体过程。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例中涉及的序列如下:
LL-37的DNA序列:
ctgctgggtgatttcttccggaaatctaaagagaagattggcaaagagtttaaaagaattgtccagagaatcaag gattttttgcggaatcttgtacccaggacagagtcc
LL-37的氨基酸序列:
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
TM的DNA序列:
gccgagcagagagccagccctctgacgtccatcgtctctgcggtggttggcattctgctggtcgtggtcttggg ggtggtctttgggatcctcatcaagcgacggcagcagaagatccggaagtacacg
TM的氨基酸序列:
AEQRASPLTSIVSAVVGILLVVVLGVVFGILIKRRQQKIRKYT
实施例1装载LL-37外泌体的设计与构建
分别在编码LL-37成熟肽的DNA序列的5’端和3’端连接上跨膜TM序列,将载体从EcoRI和SalI位点进行酶切,将融合后的序列***到酶切后的携带有EGFP的空载体中构建表达TM-LL-37和LL-37-TM的融合质粒。将TM-LL-37质粒、LL-37-TM质粒以及对照vector质粒用PEI转染试剂转染到293T细胞中,48h后收细胞培养上清,将上清先进行2000×g,15min低速离心,10000×g,30min高速离心,最后经过0.22μm滤膜过滤到超离管中进行超速离心(31000rpm,1h 30min),将超离管中的沉淀用PBS重悬,获得装载LL-37的外泌体。
将TM-LL-37质粒、LL-37-TM质粒以及对照vector质粒用PEI转染试剂转染到293T细胞中,结果如图3所示,由于质粒中带有EGFP,转染48h后通过在荧光显微镜下观察细胞中的荧光情况判断有无表达以及Western Blotting验证质粒过表达;如图4所示,将分离纯化后的外泌体进行Western Blotting,在外泌体中检测到了LL-37以及外泌体的标志蛋白,如:TSG-101、Alix、CD9,证明获得的是外泌体且设计的装载LL-37的外泌体体系构建成功;如图5所示,将vector-exo、TM-LL-37-exo、LL-37-TM-exo进行NTA分析,结果表明,对外泌体进行工程化修饰后,对其粒径变化没有明显影响;为了对装载在外泌体中的LL-37进行定量,将20μg LL-37-TM-exo与0.125μg、0.25μg、0.5μg和1.0μg的LL-37一起跑电泳,如图6所示,Western Blotting结果显示:100μg的LL-37-TM-exo中大约含有2.5μg的LL-37。
实施例2装载LL-37的外泌体的体外安全性评估
为了确定装载LL-37的外泌体使用的安全浓度,检测了vector-exo、TM-LL-37-exo、LL-37-TM-exo对Vero E6细胞、HeLa细胞、A549细胞、U251细胞、293T细胞活力的影响。将处于对数生长期的Vero E6细胞、HeLa细胞、A549细胞、U251细胞、293T细胞均匀铺于96孔板,每孔细胞数约为1×104个,于37℃培养箱中培养至细胞完全贴壁,将vector-exo、TM-LL-37-exo、LL-37-TM-exo分别用PBS按二倍梯度稀释后,加入每孔,每孔的终浓度分别为2.5μg/mL、5.0μg/mL、10.0μg/mL,阴性对照孔加入等体积的PBS,轻轻混匀,每个浓度设置六个复孔,37℃培养48h后弃去培养基,配制含10%CCK8的无血清的DMEM培养基,以换液形式每孔加入100μL,10-20min后于450nm处检测每孔的吸光度值。
结果如图7所示,vector-exo、TM-LL-37-exo、LL-37-TM-exo在≤10.0μg/mL的浓度范围内,对Vero E6细胞的细胞活力没有影响;LL-37-TM-exo在≤20.0μg/mL的浓度范围内,对HeLa细胞、A549细胞、U251细胞、293T细胞的活力均没有影响。因此,在后续的体外实验中,vector-exo、TM-LL-37-exo、LL-37-TM-exo在Vero细胞中的使用浓度设置为≤10.0μg/mL,在HeLa细胞、A549细胞、U251细胞、293T细胞中的使用浓度设置为≤20.0μg/mL。
实施例3装载LL-37的外泌体在体外可以显著抑制ZIKV的感染,而且LL-37-TM-exo比TM-LL-37-exo的抗病毒效果更显著
将处于对数生长期的Vero E6细胞均匀铺于24孔板,每孔细胞数约为1×105个,于37℃培养箱中培养至细胞完全贴壁,加入ZIKV感染(MOI=1),然后将1.25、2.5、5.0、10.0μg/mL的vector-exo、TM-LL-37-exo、LL-37-TM-exo分别加入每孔,并以PBS作为对照,轻轻混匀,37℃培养2h后,弃掉培养基,加入2%FBS的DMEM并补加相同剂量的vector-exo、TM-LL-37-exo、LL-37-TM-exo,继续培养48h后,用Trizol提取细胞内总RNA,然后用实时荧光定量PCR检测细胞中病毒RNA的合成水平。
结果如图8所示:TM-LL-37-exo、LL-37-TM-exo在体外可以显著抑制ZIKV的RNA的合成并具有剂量依赖效应。
为了进一步证明装载LL-37的外泌体可以抑制ZIKV感染,又检测了TM-LL-37-exo、LL-37-TM-exo对病毒非结构蛋白NS3和E蛋白表达的影响。将处于对数生长期的Vero E6细胞均匀铺于24孔板,每孔细胞数约为1×105个,于37℃培养箱中培养至细胞完全贴壁,加入ZIKV感染(MOI=1),然后将1.25、2.5、5.0、10.0μg/mL的vector-exo、TM-LL-37-exo、LL-37-TM-exo分别加入每孔,并以PBS作为对照,轻轻混匀,37℃培养2h后,弃掉培养基,加入2%FBS的DMEM并补加相同剂量的vector-exo、TM-LL-37-exo、LL-37-TM-exo,继续培养48h后,采用Western Blotting检测病毒非结构蛋白NS3的表达水平、免疫荧光检测病毒E蛋白的表达水平。
结果如图9所示:TM-LL-37-exo、LL-37-TM-exo在体外能显著抑制病毒NS3蛋白的表达,并且有剂量依赖效应。
实施例4在体内,LL-37-TM-exo比TM-LL-37-exo的抗ZIKV效果也更显著
将C57BL/6J小鼠分为3组(vector-exo、TM-LL-37-exo、LL-37-TM-exo),以106PFU的ZIKV经尾静脉感染小鼠,1h后尾静脉注射vector-exo以及10μg/只、20μg/只、40μg/只的TM-LL-37-exo和LL-37-TM-exo。4天后取小鼠的脑、睾丸、肝脏,通过Q-PCR检测TM-LL-37-exo、LL-37-TM-exo在小鼠体内对ZIKV感染的作用。
结果如图10所示,装载LL-37的外泌体能够显著降低C57BL/6J雄鼠肝、脑、睾丸组织中的病毒载量,且与体外实验结果一致,LL-37-TM-exo的抗病毒效果更显著,且在10μg时就效果显著。
实施例5外泌体装载的LL-37比LL-37的血清学稳定性高
将处于对数生长期的Vero E6细胞铺于24孔板于37℃培养箱中培养,每孔细胞数约为5×104个,待细胞密度达到70-80%时,弃去培养基,PBS洗涤三次,每孔加入ZIKV,使得MOI(感染复数Multiplicity ofinfection)=1,同时分别加入已经在37℃孵育不同时间后的LL-37-TM-exo和PBS、LL-37-TM-exo和serum、LL-37和PBS、LL-37和serum的孵育混合物,并以PBS作为对照,37℃培养2h后,弃掉培养基,加入2%FBS的DMEM,继续培养48h后,通过Western Blotting对病毒复制动力学进行检测,从而比较二者之间的血清稳定性。
具体实验操作过程见图15。结果如图11所示,与血清共孵育1h时,LL-37和LL-37-TM-exo的抗ZIKV活性均没有受到影响;当与血清共孵育6h时,LL-37的抗病毒能力显著下降,LL-37-TM-exo依然对ZIKV感染具有良好地抵抗作用。
实施例5LL-37-TM-exo在C57BL/6J中对ZIKV感染具有预防作用
将C57BL/6J小鼠分为2组(vector-exo组、LL-37-TM-exo组),各组分别尾静脉注射vector-exo、LL-37-TM-exo,1h后以106PFU的ZIKV经尾静脉感染小鼠。4天后取小鼠的脑、睾丸、肝脏、脾脏,通过Q-PCR检测LL-37-TM-exo在小鼠体内对ZIKV感染的预防作用。
结果如图12所示,LL-37-TM-exo能够显著降低C57BL/6J的病毒载量。
实施例6LL-37-TM-exo在C57BL/6J中对ZIKV感染具有治疗作用
将C57BL/6J小鼠分为2组(vector-exo组、LL-37-TM-exo组),以106PFU的ZIKV经尾静脉感染小鼠,1h后各组分别尾静脉注射vector-exo、LL-37-TM-exo。4天后取小鼠的脑、睾丸、肝脏,通过Q-PCR检测LL-37-TM-exo在小鼠体内对ZIKV感染的作用。
结果如图13所示:LL-37-TM-exo能够显著降低C57BL/6J雄鼠肝、脑、睾丸组织中的病毒载量。
实施例7LL-37-TM-exo在C57BL/6J小鼠体内的抗病毒活性比LL-37大大提高
将C57BL/6J小鼠分为4组(PBS组、vector-exo组、LL-37-TM-exo组和LL-37组),以106PFU的ZIKV经尾静脉感染小鼠,1h后各组分别尾静脉注射PBS、vector-exo(10μg/只,0.5mg/kg)、LL-37-TM-exo(10μg/只,0.5mg/kg)和LL-37(80μg/只,4mg/kg)。4天后取小鼠的脑、睾丸、脾脏,通过Q-PCR检测LL-37-TM-exo在小鼠体内对ZIKV感染的作用。
结果如图14所示,每只小鼠注射10μg(0.5mg/kg)的LL-37-TM-exo对ZIKV复制的抑制效果相当于、甚至优于给每只小鼠注射80μg的LL-37(4mg/kg)。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (5)
1.一种装载抗菌肽LL-37的外泌体,其特征在于:所述抗菌肽LL-37通过修饰在其N端或C端的跨膜蛋白装载于外泌体膜上,所述跨膜蛋白的氨基酸序列如SEQ ID NO.1所示,所述抗菌肽LL-37的氨基酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的装载抗菌肽LL-37的外泌体,其特征在于,所述装载抗菌肽LL-37的外泌体由以下步骤制备得到:将跨膜蛋白编码基因融合至抗菌肽LL-37编码基因的5’端或3’端,再与表达载体连接得到重组质粒,将重组质粒转入宿主细胞中,构建得到重组细胞,并将该重组细胞通过差速离心法、TFF切向流过滤法、密度梯度离心、空间排阻色谱法、聚合物沉淀法或免疫亲和法制备得到装载抗菌肽LL-37的外泌体。
3.权利要求1或2所述的装载抗菌肽LL-37的外泌体在制备抗寨卡病毒制剂中的应用。
4.根据权利要求3所述的应用,其特征在于:所述抗寨卡病毒制剂用于预防或治疗寨卡病毒感染。
5.一种抗寨卡病毒的药物,其特征在于:所述药物包括权利要求1或2所述的装载抗菌肽LL-37的外泌体。
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