CN113144210A - 抗菌多肽复合物及其制备方法和应用 - Google Patents
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Abstract
一种抗菌多肽复合物，为MSN@T7E21R‑HD5@SC，其中包括抗菌衍生肽T7E21R‑HD5，T7E21R‑HD5的氨基酸序列为ATCYCRRGRCATRESLSGVCRISGRLYRLCCR(Cys3‑Cys31/Cys5‑Cys20/Cys10‑Cys30)。E21R突变可增强HD5的抗菌性，T7R突变不仅可以进一步强化E21R‑HD5的抗菌活性，还能恢复单体肽的聚合作用，致使T7E21R‑HD5突变肽在高浓度盐溶液和血清中依然保持高效抗菌性，MSN负载可以保护进入介孔里面的多肽，而SCN包裹MSN可以起到肠道定向释放的功能，利于药物的有效吸收，能够充分发挥HD5的抗HPV作用，有效防治HPV感染，可用于预防或治疗人乳头瘤病毒。
Description
技术领域
本发明涉及抗体药物的技术领域，更具体地说涉及一种抗菌多肽复合物及其制备方法和应用。
背景技术
人乳头瘤病毒（HPV）已被确定为全球约5％的癌症的原因。HPV感染几乎与所有宫颈癌和肛门生殖器（外阴，阴道，阴茎和肛门）和口咽癌相关其高风险类型包括HPV16,18,31,33,35,39,45,51,52,56,58等。持续性感染高危型HPV可明确预测女性患宫颈癌的风险。但感染高危型人乳头瘤病毒（HR-HPV）是宫颈癌的必要但不充分的原因，还需有驱动细胞恶性转化的额外病毒和宿主的遗传事件。HPV生命周期始于感染宫颈上皮细胞基底层。在正常的病毒的生命周期中，HPV基因组以游离的状态存在，并被认为在鳞状上皮的基底细胞中以每细胞约50-100拷贝的形式保留。病毒致癌基因E6和E7的表达是被严格调控，高水平的表达致癌基因E6、E7仅上皮细胞分化后。病毒致癌基因可诱导细胞周期不定期重新进入S期，激活宿主复制机制，在病毒粒子合成之前扩增病毒基因组。此时病毒癌基因的表达不会造成致癌威胁，因为这些细胞可以从宫颈鳞状上皮细胞中丢失。宫颈瘤变进展需要空间和量化上对这种严格调控转录的解除，使得病毒癌基因在整个上皮高水平表达。致癌基因长时间被保留在宫颈上皮内，因此可代表致癌因素靶细胞，其可诱导染色体高度不稳定性以驱动朝向恶性发展。
人α防御素-5（HD5），主要由肠道Paneth细胞和女性生殖道粘膜上皮细胞表达和分泌，另外在尿道上皮组织中也检测到的HD5的存在。正是由于这些区域经常受到外来微生物的侵入，环境选择导致的进化压力造就了HD5具有突出的抗病原微生物活性，因此更具开发应用价值。
发明内容
本发明克服了现有技术中的不足，提供了一种抗菌多肽复合物及其制备方法和应用。
本发明的目的通过下述技术方案予以实现。
本发明的抗菌多肽复合物，为MSN@T7E21R-HD5@SC，其中包括抗菌衍生肽T7E21R-HD5，T7E21R-HD5的氨基酸序列为ATCYCRRGRCATRESLSGVCRISGRLYRLCCR (Cys3-Cys31/Cys5-Cys20/Cys10-Cys30)，含有三对二硫键，带7个正电荷，其分子量为 3664.0 Da；6个半胱氨酸两两配对形成三对定向搭配的分子间二硫键；介孔二氧化硅纳米粒子MSN通过静电吸附作用负载T7E21R-HD5抗菌肽，琥珀酰酪蛋白SCN包裹在介孔二氧化硅纳米粒子MSNs之外。
本发明的抗菌多肽复合物的制备方法，包括以下步骤：
a.称取0.068g三乙醇胺TEA溶于25mL去离子水中，80℃搅拌30min；
b.加入0.38g溴化十六烷基三甲胺CTAB继续搅拌1h；
c.向上述溶液中加入4mL正硅酸乙酯TEOS搅拌2h；
d.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次；
e.样品加入到含1%硝酸铵的乙醇80℃回流40min，以去除CTAB；
f.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次，真空干燥4℃
保存备用；
g.取100μL浓度为1mg/mL的T7E21R-HD5溶液加到含20μgMSN的溶液中，4℃震荡孵育过夜；
h．分别将琥珀酰胺和酪氨酸充分溶解，然后将琥珀酰胺溶液滴加到酪氨酸溶液中，搅拌1h后，以氢氧化钠溶液调整混合溶液的pH至9，继续搅拌1h，得琥珀酰酪氨酸；
i.将制备好的琥珀酰化的酪氨酸滴加到MSN@T7E21R-HD5溶液中，4℃反应过夜；
j.10000rpm离心，10min。用灭菌水清洗三次，得制备好的MSN@T7E21R-HD5@SCN。
MSN与T7E21R-HD5 质量比为 2:1。
本发明的抗菌多肽复合物在预防或治疗人乳头瘤病毒HPV中的应用。
本发明的有益效果为：本发明中E21R突变可增强HD5的抗菌性，T7R突变不仅可以进一步强化E21R-HD5的抗菌活性，还能恢复单体肽的聚合作用，致使T7E21R-HD5突变肽在高浓度盐溶液和血清中依然保持高效抗菌性，MSN负载可以保护进入介孔里面的多肽，而SCN包裹MSN可以起到肠道定向释放的功能，利于药物的有效吸收，能够充分发挥HD5的抗HPV作用，有效防治HPV感染。
附图说明
图1为T7E21R-HD5的色谱检测结果示意图；
图2为不同MSN/T7E21R-HD5质量比情况下T7E21R-HD5在MSN上的负载率示意图；
图3为TEM显示MSN@T7E21R-HD5形态示意图；
图4为TEM显示经1%磷钨酸水溶液负染后的MSN@T7E21R-HD5@SCN示意图；
图5为SDS电泳验证MSN@T7E21R-HD5@SCN制备成功示意图；
图6为抗菌多肽复合物不同浓度作用下HPV16对C33a细胞的48h后GFP表达率示意图；
图7为抗菌多肽复合物对HPV16感染的抑制率曲线示意图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的说明。
本发明的抗菌多肽复合物，为MSN@T7E21R-HD5@SC，其中包括抗菌衍生肽T7E21R-HD5，T7E21R-HD5的氨基酸序列为ATCYCRRGRCATRESLSGVCRISGRLYRLCCR (Cys3-Cys31/Cys5-Cys20/Cys10-Cys30)，含有三对二硫键，带7个正电荷，其分子量为 3664.0 Da；6个半胱氨酸两两配对形成三对定向搭配的分子间二硫键；介孔二氧化硅纳米粒子MSN通过静电吸附作用负载T7E21R-HD5抗菌肽，琥珀酰酪蛋白SCN包裹在介孔二氧化硅纳米粒子MSNs之外。
本发明的抗菌多肽复合物的制备方法，包括以下步骤：
a.称取0.068g三乙醇胺TEA溶于25mL去离子水中，80℃搅拌30min；
b.加入0.38g溴化十六烷基三甲胺CTAB继续搅拌1h；
c.向上述溶液中加入4mL正硅酸乙酯TEOS搅拌2h；
d.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次；
e.样品加入到含1%硝酸铵的乙醇80℃回流40min，以去除CTAB；
f.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次，真空干燥4℃
保存备用；
g.取100μL浓度为1mg/mL的T7E21R-HD5溶液加到含20μgMSN的溶液中，4℃震荡孵育过夜；
h．分别将琥珀酰胺和酪氨酸充分溶解，然后将琥珀酰胺溶液滴加到酪氨酸溶液中，搅拌1h后，以氢氧化钠溶液调整混合溶液的pH至9，继续搅拌1h，得琥珀酰酪氨酸；
i.将制备好的琥珀酰化的酪氨酸滴加到MSN@T7E21R-HD5溶液中，4℃反应过夜；
j.10000rpm离心，10min。用灭菌水清洗三次，得制备好的MSN@T7E21R-HD5@SCN。
MSN与T7E21R-HD5质量比为2:1，T7E21R-HD5的负载率最大，可达到70%。
T7E21R-HD5负载后纳米粒的形态、粒径大小基本无变化，但与MSN相比，MSN@T7E21R-HD5分散性较差，趋于聚集，介孔由于多肽负载而变模糊。
MSN的Zeta电位为-14.6±0.4，MSN@T7E21R-HD5的Zeta电位为+14±0.4，电位反转提示T7E21R-HD5的成功负载。
在前期MSN@T7E21R-HD5基础上制备MSN@T7E21R-HD5@SCN，MSN@T7E21R-HD5@SCN制备后以1%磷钨酸水溶液负染，透射电镜观察，纳米材料外面包裹一层深色物质即为SCN。嵌入图为未用1%磷钨酸水溶液负染的MSN@T7E21R-HD5@SCN的透射电镜图，可见纳米粒周围有一圈颜色偏浅的色带SCN围绕，检测MSN@T7E21R-HD5@SCN的zeta电位和粒径，发现其Zeta电位变为-23.6，水合粒径较MSN@T7E21R-HD5增大，提示MSN@T7E21R-HD5@SCN制备成功。
经MSN、T7E21R-HD5、SCN、MSN@T7E21R-HD5@SCN样品变性后SDS电泳，确认MSN@T7E21R-HD5@SCN中含有T7E21R-HD5及SCN，证明复合纳米粒制备成功。
将浓度梯度稀释的本发明的抗菌多肽复合物分别与HPV16病毒（浓度2×106）加入细胞培养体系，继续培养之后，荧光显微镜下观察和计数。由图可见，随着多肽浓度的加大，细胞阳性表达率逐步减少，当浓度达到50μg/ml时，镜下已经观察不到细胞的表达。
进一步进行流式细胞术检测后，根据测结果即GFP阳性细胞计数绘制出抗菌多肽复合物的抗HPV16感染作用量效曲线和对HPV16感染的抑制率曲线。结果表明抗菌多肽复合物对HPV16感染具有显著的抑制作用，并呈现出明显的剂量效应关系，当浓度为2μg/ml时已可以观察到对病毒的抑制作用；多肽浓度达到50μg/ml时，对感染的抑制率已接近100%。
本发明的抗菌多肽复合物在预防或治疗人乳头瘤病毒HPV中的应用。
以上对本发明进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Claims (4)
1.一种抗菌多肽复合物，为MSN@T7E21R-HD5@SC，其中包括抗菌衍生肽T7E21R-HD5，T7E21R-HD5的氨基酸序列为ATCYCRRGRCATRESLSGVCRISGRLYRLCCR (Cys3-Cys31/Cys5-Cys20/Cys10-Cys30)，含有三对二硫键，带7个正电荷，其分子量为 3664.0 Da；6个半胱氨酸两两配对形成三对定向搭配的分子间二硫键；介孔二氧化硅纳米粒子MSN通过静电吸附作用负载T7E21R-HD5抗菌肽，琥珀酰酪蛋白SCN包裹在介孔二氧化硅纳米粒子MSNs之外。
2.一种权利要求1所述抗菌多肽复合物的制备方法，包括以下步骤：
a.称取0.068g三乙醇胺TEA溶于25mL去离子水中，80℃搅拌30min；
b.加入0.38g溴化十六烷基三甲胺CTAB继续搅拌1h；
c.向上述溶液中加入4mL正硅酸乙酯TEOS搅拌2h；
d.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次；
e.样品加入到含1%硝酸铵的乙醇80℃回流40min，以去除CTAB；
f.12000rpm离心15min收集反应产物，去离子水和乙醇各洗三次，真空干燥4℃
保存备用；
g.取100μL浓度为1mg/mL的T7E21R-HD5溶液加到含20μgMSN的溶液中，4℃震荡孵育过夜；
h．分别将琥珀酰胺和酪氨酸充分溶解，然后将琥珀酰胺溶液滴加到酪氨酸溶液中，搅拌1h后，以氢氧化钠溶液调整混合溶液的pH至9，继续搅拌1h，得琥珀酰酪氨酸；
i.将制备好的琥珀酰化的酪氨酸滴加到MSN@T7E21R-HD5溶液中，4℃反应过夜；
j.10000rpm离心，10min，
用灭菌水清洗三次，得制备好的MSN@T7E21R-HD5@SCN。
3.根据权利要求2所述的所述抗菌多肽复合物的制备方法，其特征在于：MSN与T7E21R-HD5 质量比为 2:1。
4.一种权利要求1所述抗菌多肽复合物在预防或治疗人乳头瘤病毒HPV中的应用。
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| CN111375051A (zh) * | 2018-12-29 | 2020-07-07 | 江苏吉锐生物技术有限公司 | 一种多肽在制备预防和治疗人乳头瘤病毒感染制剂的应用 |
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