CN114404614B - 一种靶向铜绿假单胞菌的免疫脂质体及其制备方法和应用 - Google Patents
一种靶向铜绿假单胞菌的免疫脂质体及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术药物领域,尤其涉及一种靶向铜绿假单胞菌的免疫脂质体及其制备方法和应用。本发明的免疫脂质体由单克隆抗体、治疗药物和纳米脂质体组成,以抗铜绿假单胞菌的单克隆抗体为靶向剂,以环丙沙星为治疗药物,以纳米脂质体为载体。本发明提供的免疫脂质体可以赋予广谱抗生素靶向性,将抗生素富集到细菌附近,提高感染部位的局部药物浓度,避免细菌耐药性的产生。本发明提供的免疫脂质体能够减少抗生素的用药剂量,提高感染性疾病的疗效。
Description
技术领域
本发明涉及生物技术药物领域,特别是一种靶向铜绿假单胞菌的免疫脂质体及其制备方法和应用。
背景技术
铜绿假单胞菌(Pseudomonas aeruginosa)是一种常见的革兰氏阴性杆状细菌,能够引起植物及动物包括人类的感染,临床上铜绿假单胞菌感染是患有癌症,囊性纤维化和烧伤等患者的严重并发症之一,死亡率高。除此之外,铜绿假单胞菌还能引起多种其他感染,包括心内膜炎、肺炎和泌尿道感染,以及中枢神经系统、伤口、眼睛、耳朵、皮肤和肌肉骨骼系统感染等。铜绿假单胞菌是医院感染的主要原因,每年影响200多万患者,每年造成约9万人死亡。铜绿假单胞菌的耐药机制包括表达抗生素失活酶、外排系统、入侵宿主细胞形成胞内菌、形成生物被膜等。
针对抗生素耐药铜绿假单胞菌,研究人员也一直在积极寻找新型的抗生素替代疗法,包括阻断或中和毒素产生、调整菌群失调、应用疫苗或抗体、抗微生物多肽及采用具有免疫调节功能的细胞因子等。在所有新型疗法中,单克隆抗体对单一种类的细菌具有很高的靶标特异性,因此不会破坏微生物群,具有安全高效的特点。脂质体是研究最广泛的纳米制剂之一,具有良好的生物相容性和可修饰性,在药物递送方面有广泛的应用。
免疫脂质体即在纳米脂质体表面修饰具有靶向作用的靶向剂,如单克隆抗体。免疫脂质体能选择性的将包埋的药物浓集定位于靶组织、靶器官或靶细胞;相比普通脂质体,免疫脂质体具有毒性更低,靶向性更强等优点。结合单克隆抗体的靶向性,以脂质体作为抗生素药物载体,可以有效提高感染部位抗生素的局部浓度,避免细菌耐药性的产生,并有利于减小全身给药剂量,降低毒副作用。免疫脂质体有望成为对抗多药耐药菌感染等严重性感染性疾病的有效治疗策略。
发明内容
针对现有技术的不足,本发明提供一种同时载荷抗铜绿假单胞菌药物和抗铜绿假单胞菌单克隆抗体的免疫脂质体来解决现有药物治疗铜绿假单胞菌所致的严重感染性疾病毒副作用大、容易产生细菌耐药性的问题。
为达到此目的,本发明采用以下技术方案:
本发明第一方面提供一种靶向铜绿假单胞菌的免疫脂质体,所述免疫脂质体包括抗铜绿假单胞菌单克隆抗体、抗铜绿假单胞菌药物和纳米脂质体;通过负载抗铜绿假单胞菌药物的纳米脂质体与抗铜绿假单胞菌单克隆抗体共价偶联形成。
进一步地,所述抗铜绿假单胞菌单克隆抗体为靶向铜绿假单胞菌PcrV蛋白的单克隆抗体。优选地,所述抗铜绿假单胞菌单克隆抗体为靶向铜绿假单胞菌(III型分泌系统)的PcrV蛋白的单克隆抗体1F3HL。
进一步地,所述抗铜绿假单胞菌药物为环丙沙星。
进一步地,所述纳米脂质体包括磷脂、胆固醇(CHOL)和辅料。
在本发明的一些实施例中,所述负载抗铜绿假单胞菌药物的纳米脂质体中药物浓度为1~5mg/mL。
在本发明的一些实施例中,所述免疫脂质体的平均粒径为100~200nm。
在本发明的一些实施例中,所述免疫脂质体的抗铜绿假单胞菌药物包封率大于80%。
在本发明的一些实施例中,所述免疫脂质体中抗铜绿假单胞菌单克隆抗体的偶联率大于95%。
在本发明的一些实施例中,所述环丙沙星与纳米脂质体中磷脂的摩尔比为0.1~0.5:1,在该比例下,可实现环丙沙星包封率>80%。
在本发明的一些实施例中,所述抗铜绿假单胞菌单克隆抗体与纳米脂质体中磷脂含量的摩尔比为1:100~1000。
在本发明的一些实施例中,所述磷脂选用氢化大豆卵磷脂(HSPC)。
在本发明的一些实施例中,所述辅料选用二硬脂酰磷脂酰乙醇胺–聚乙二醇(DSPE-PEG),优选为DSPE-PEG2000;对脂质体表面进行聚乙二醇(PEG)修饰后可以延长脂质体的半衰期和提高其在血液循环中的稳定性、改变脂质体的生物学分布,DSPE-PEG2000的长循环效果较好。
在本发明的一些实施例中,所述纳米脂质体中HSPC与CHOL的质量比为0.1~1.0:1。
在本发明的一些实施例中,所述纳米脂质体中HSPC与DSPE-PEG2000的质量比为0.1~1.0:1。
本发明第二方面提供一种靶向铜绿假单胞菌的免疫脂质体的制备方法,所述制备方法包括如下步骤:
步骤一、将空白脂质体与抗铜绿假单胞菌药物混合孵育,得到载药纳米脂质体;
步骤二、将抗铜绿假单胞菌单克隆抗体与连接脂质混合反应,得到单克隆抗体脂质共聚物;
步骤三、将步骤一所得载药纳米脂质体与步骤二所得单克隆抗体脂质共聚物共孵育,得到靶向铜绿假单胞菌的免疫脂质体。
在本发明的一些实施例中,步骤一中所述混合孵育时间为20~40min。
在本发明的一些实施例中,步骤一中混合孵育后还需用10%蔗糖溶液透析,去除游离的抗铜绿假单胞菌药物。
在本发明的一些实施例中,步骤二中所述连接脂质为DSPE-PEG2000-NHS。
在本发明的一些实施例中,步骤二中所述混合反应具体为4℃混合过夜反应。
在本发明的一些实施例中,步骤二中混合反应后还需透析去除未反应的DSPE-PEG2000-NHS。
在本发明的一些实施例中,步骤三中共孵育后还需用10mM羟乙基哌嗪乙硫磺酸(HEPES)缓冲液透析纯化,去除未插入的抗铜绿假单胞菌单克隆抗体及单克隆抗体脂质共聚物。
在本发明的一些实施例中,步骤一中所述空白脂质体的制备方法包括以下步骤:
S1、先将胆固醇溶于乙醇,然后按质量配比加入磷脂和辅料,待溶解后加入硫酸铵溶液,进行水合反应,得到水合产物;
S2、将步骤S1所得的水合产物通过脂质体挤出仪制备得到脂质体混悬液;
S3、将步骤S2所得脂质体混悬液透析,得到空白脂质体。
进一步地,步骤S1中所述磷脂与胆固醇的质量比为0.1~1.0:1。
进一步地,步骤S1中所述磷脂与所述辅料的质量比为0.1~1.0:1。
进一步地,步骤S1中所述水合反应时间为20~40min。
进一步地,步骤S1中所述硫酸铵溶液的pH为5.0~6.0,优选pH为5.5。
进一步地,步骤S3中透析所用的溶液为10%蔗糖溶液。
在本发明的一些实施例中,步骤二中所述抗铜绿假单胞菌单克隆抗体的制备方法包括以下步骤:
T1、构建包含所述单克隆抗体的编码基因的表达载体;
T2、通过瞬时转染宿主细胞的方法构建包含所述表达载体的宿主细胞;
T3、连续培养所述宿主细胞并收集细胞培养上清液;
T4、进行目的蛋白纯化,得到抗铜绿假单胞菌单克隆抗体。
进一步地,步骤T1中所述单克隆抗体为靶向铜绿假单胞菌PcrV蛋白的单克隆抗体。
进一步地,步骤T1中所述表达载体为pM09表达载体。
进一步地,步骤T2中所述宿主细胞为人HEK293E细胞。
进一步地,步骤T4中所述纯化为亲和层析柱纯化;优选地,所述亲和层析柱的填料为ProteinA。
进一步地,所述步骤T3中所述收集细胞培养上清液的时间为连续培养细胞6~7天后。
本发明采用乙醇注入法制备了纳米脂质体,采用硫酸铵梯度法制备载药纳米脂质体,采用后插入法将抗铜绿假单胞菌的单克隆抗体修饰到载药纳米脂质体表面,制备得到免疫脂质体。
本发明第三方面提供一种上述靶向铜绿假单胞菌的免疫脂质体在制备治疗铜绿假单胞菌感染药物和递送工具中的应用。
本发明第四方面提供一种上述靶向铜绿假单胞菌的免疫脂质体在制备治疗多药耐药性铜绿假单胞菌感染药物中的应用。
本发明与现有技术相比,具有以下有益效果:
1、本发明提供的靶向铜绿假单胞菌的免疫脂质体赋予广谱抗生素靶向性,高效地将抗生素富集到细菌附近,以此提高感染部位的局部药物浓度,避免细菌耐药性的产生,并有利于减小全身给药量,降低毒副作用,提高感染性疾病疗效。
2、本发明提供的靶向铜绿假单胞菌的免疫脂质体,一方面,其平均粒径为100~200nm,可以通过被动靶向方式,将抗菌药物有效地输送到炎症组织;另一方面,其抗体偶联率大于95%,可以通过特异性抗体对细菌进行主动靶向,提高感染部位的局部药物浓度。
3、本发明提供的靶向铜绿假单胞菌的免疫脂质体对抗铜绿假单胞菌药物的包封率大于80%,能够实现较高浓度的抗菌药物靶向递送,且结构稳定,生物相容性好,抗菌活性优于相同浓度的抗铜绿假单胞菌药物溶液,在治疗多药耐药性铜绿假单胞菌感染等严重感染性疾病方面具有良好的应用前景。
附图说明
图1本发明所述免疫脂质体的制备技术路线及其结构示意图。
图2本发明所述单克隆抗体ProteinA亲和层析柱亲和纯化图。
图3本发明所述单克隆抗体经亲和层析后SDS-PAGE图。
图4本发明所述单克隆抗体与抗原Biacore检测结果图。
图5本发明实验确定环丙沙星最大吸收波长结果图。
图6本发明所述免疫脂质体SDS-PAGE图。
图7本发明所述免疫脂质体体外抑菌效果图。
具体实施方式
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
以下实施例以环丙沙星作为抗铜绿假单胞菌药物,采用乙醇注入法制备空白脂质体,然后采用硫酸铵梯度法制备得到载药纳米脂质体,最后采用后插入法将靶向铜绿假单胞菌T3SS(III型分泌系统)的PcrV蛋白的单克隆抗体1F3HL修饰到载药纳米脂质体表面,制备得到靶向铜绿假单胞菌的免疫脂质体。
以下实施例中所用到的载体、基因及菌种等来源说明:
载体来源为实验室保存;
引物由上海睿勉基因科技有限公司合成;
菌种来源实验室保存,为ATCC27853铜绿假单胞菌。
实施例1:单克隆抗体的制备及表征
本发明通过重组表达获得抗铜绿假单胞菌单克隆抗体。具体地说,将该单克隆抗体的基因构建于真核表达载体中,以人胚肾HEK293E细胞为表达宿主,通过PEI法瞬时转染。培养适当时间后,收集细胞培养上清液,使用Protein A柱进行亲和层析纯化。纯化产物经SDS-PAGE检测纯度,最终通过BCA蛋白定量法定量。具体操作步骤如下:
(1)载体质粒获取:抗铜绿假单胞菌单克隆抗体(1F3HL)的轻链(LC)氨基酸序列如SEQ ID NO:1所示,重链(HC)氨基酸序列如SEQ ID NO:2所示,设计引物进行常规PCR扩增片段,引物序列见下表1,并将其插入pM09质粒载体;最终构建的质粒载体通过测序验证并进行大量提取。
表1:抗铜绿假单胞菌单克隆抗体(1F3HL)PCR扩增所用引物序列
(2)以人胚肾HEK293E细胞为表达宿主,采用PEI法进行瞬时转染。
(3)连续培养细胞,监测细胞存活率变化,约6-7天后收集细胞培养上清液,使用AKTA STAR纯化系统、ProteinA亲和层析柱进行目的蛋白纯化;蛋白纯化图谱见图2。
(4)纯化所得蛋白产物,通过超滤法将蛋白溶剂置换为PBS,随后通过SDS-PAGE鉴定其纯度,结果见图3,其中Reduced为经还原处理的样品,Non-reduced为未经还原处理的样品。
采用Biacore 8K,CM5芯片测定上述表达纯化所得单克隆抗体与铜绿假单胞菌表面抗原蛋白PcrV的亲和力,实验结果见表2和图4,计算得单克隆抗体1F3HL与PcrV抗原蛋白的KD=2.96E-8M。
表2:1F3HL与PcrV的SPR数据结果
实施例2:免疫脂质体的制备
2.1、载环丙沙星纳米脂质体的制备
(1)分别精密称取处方量的HSPC,CHOL和DSPE-PEG2000。
(2)先将CHOL溶于无水乙醇;随后加入HSPC和DSPE-PEG2000,待其溶解;再加入硫酸铵溶液(pH5.5),水合30min。
(3)利用脂质体挤出仪,将水合好的脂质体样品通过80-200nm的聚碳酸酯膜,每个层级推挤6-10次,即可得脂质体混悬液;将所得脂质体混悬液用10%蔗糖溶液透析,制备得到空白脂质体。
(4)将环丙沙星溶液与空白脂质体共孵育30min,再次用10%蔗糖溶液透析,去除游离的药物,即获得载环丙沙星纳米脂质体。
2.2、免疫脂质体的制备
(1)制备单克隆抗体脂质共聚物:将抗铜绿假单胞菌单克隆抗体与连接脂质DSPE-PEG2000-NHS于4℃混合过夜反应,将反应后的DSPE-PEG-mAb透析去除未反应的DSPE-PEG2000-NHS,得到单克隆抗体脂质共聚物。
(2)将单克隆抗体脂质共聚物与2.1制得的载环丙沙星纳米脂质体共孵育,即可将抗体后插入到载环丙沙星免疫脂质体上,再用10mM HEPES缓冲液透析纯化,去除未插入的单克隆抗体及单克隆抗体脂质共聚物,即可获得所述免疫脂质体。
实施例3:免疫脂质体的表征及生物学功能研究
3.1免疫脂质体的表征
3.1.1粒径测定:免疫脂质体悬液适量以蒸馏水适当稀释后于25℃下经激光粒径测定仪测定其粒径分布,靶向铜绿假单胞菌的免疫脂质体平均粒径为100-200nm。
3.1.2包封率测定:使用紫外分光光度法测定环丙沙星含量。对环丙沙星溶液进行全波长(200-800nm)扫描,以溶剂作为空白对照,结果如图5所示,表明环丙沙星的最大吸收波长为285nm。经检测得,靶向铜绿假单胞菌的免疫脂质体的环丙沙星包封率大于80%。
3.1.3验证抗体是否偶联成功:所述免疫脂质体通过聚丙烯酰胺凝胶电泳(SDSPAGE)分析,得出抗铜绿假单胞菌单克隆抗体在原始分子量的基础上有所增加(如图6所示),表明环丙沙星脂质体与抗铜绿假单胞菌单克隆抗体偶联成功。
3.1.4抗体偶联率测定:所述免疫脂质体通过BCA蛋白定量法分析。测定环丙沙星脂质体与抗体偶联并经透析纯化后,免疫脂质体混悬液中的蛋白含量与透析纯化前蛋白含量的比值,结果表明该免疫脂质体的抗体的偶联率大于95%。
3.1.5稳定性研究:所述免疫脂质体于4℃,10mM HEPES缓冲液中存储7天内,粒径和包封率稳定,粒径大小变化<5%,包封率变化<5%。
3.2免疫脂质体体外抑菌活性的研究
以环丙沙星溶液为对照,将环丙沙星和免疫脂质体(mAb-LP)用LB细菌培养基稀释至不同浓度,接种相同量的细菌(1×106CFU/孔),于96孔板中,于37℃恒温培养箱静置培养。培养12h和24h后,通过酶标仪检测OD600,以反映细菌生长情况,如图7所示。结果表明,在低浓度环丙沙星的作用下,免疫脂质体对铜绿假单胞菌的生长一直效果明显优于环丙沙星裸药;且在延长培养时间(24h)时,免疫脂质体的抑菌效果更加明显。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
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Thr Met His Thr Val Ala Gly Ala Pro Gly Gly Gly Leu Gly Thr Met
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Gly Gly Ile Ala Pro Ser Ala Gly Ala Thr Ala Thr Ala Gly Leu Pro
50 55 60
Ala Thr Ala Val Thr Met Thr Ala Ala Thr Ser Thr Ser Thr Val Thr
65 70 75 80
Met Gly Leu Ser Ser Leu Ala Ser Gly Ala Thr Ala Val Thr Thr Cys
85 90 95
Val Leu Thr Gly Ala Thr Val Val Thr Thr Thr Met Ala Thr Thr Gly
100 105 110
Gly Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Leu Gly Pro Ser
115 120 125
Val Pro Pro Leu Ala Pro Ser Ser Leu Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Leu Ala Thr Pro Pro Gly Pro Val Thr Val
145 150 155 160
Ser Thr Ala Ser Gly Ala Leu Thr Ser Gly Val His Thr Pro Pro Ala
165 170 175
Val Leu Gly Ser Ser Gly Leu Thr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gly Thr Thr Ile Cys Ala Val Ala His
195 200 205
Leu Pro Ser Ala Thr Leu Val Ala Leu Leu Val Gly Pro Leu Ser Cys
210 215 220
Ala Leu Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Pro Leu Pro Pro Pro Leu Pro Leu Ala Thr Leu Met
245 250 255
Ile Ser Ala Thr Pro Gly Val Thr Cys Val Val Val Ala Val Ser His
260 265 270
Gly Ala Pro Gly Val Leu Pro Ala Thr Thr Val Ala Gly Val Gly Val
275 280 285
His Ala Ala Leu Thr Leu Pro Ala Gly Gly Gly Thr Ala Ser Thr Thr
290 295 300
Ala Val Val Ser Val Leu Thr Val Leu His Gly Ala Thr Leu Ala Gly
305 310 315 320
Leu Gly Thr Leu Cys Leu Val Ser Ala Leu Ala Leu Pro Ala Pro Ile
325 330 335
Gly Leu Thr Ile Ser Leu Ala Leu Gly Gly Pro Ala Gly Pro Gly Val
340 345 350
Thr Thr Leu Pro Pro Ser Ala Ala Gly Leu Thr Leu Ala Gly Val Ser
355 360 365
Leu Thr Cys Leu Val Leu Gly Pro Thr Pro Ser Ala Ile Ala Val Gly
370 375 380
Thr Gly Ser Ala Gly Gly Pro Gly Ala Ala Thr Leu Thr Thr Pro Pro
385 390 395 400
Val Leu Ala Ser Ala Gly Ser Pro Pro Leu Thr Ser Leu Leu Thr Val
405 410 415
Ala Leu Ser Ala Thr Gly Gly Gly Ala Val Pro Ser Cys Ser Val Met
420 425 430
His Gly Ala Leu His Ala His Thr Thr Gly Leu Ser Leu Ser Leu Ser
435 440 445
Pro Gly Leu
450
Claims (9)
1.一种靶向铜绿假单胞菌的免疫脂质体,其特征在于,所述免疫脂质体包括抗铜绿假单胞菌单克隆抗体、抗铜绿假单胞菌药物和纳米脂质体;通过负载抗铜绿假单胞菌药物的纳米脂质体与抗铜绿假单胞菌单克隆抗体共价偶联形成;所述抗铜绿假单胞菌单克隆抗体为靶向铜绿假单胞菌PcrV蛋白的单克隆抗体,其轻链氨基酸序列如SEQ ID NO:1所示,重链氨基酸序列如SEQ ID NO:2所示;所述抗铜绿假单胞菌药物为环丙沙星;所述免疫脂质体的平均粒径为100~200 nm;所述免疫脂质体中抗铜绿假单胞菌单克隆抗体的偶联率大于95%。
2.如权利要求1所述的靶向铜绿假单胞菌的免疫脂质体,其特征在于,还包括如下特征中的一项或几项:
(1)所述纳米脂质体包括磷脂、胆固醇和辅料;所述辅料选用二硬脂酰磷脂酰乙醇胺–聚乙二醇;
(2)所述负载抗铜绿假单胞菌药物的纳米脂质体中药物浓度为1~5 mg/mL;
(3)所述免疫脂质体的抗铜绿假单胞菌药物包封率大于80%。
3.如权利要求2所述的靶向铜绿假单胞菌的免疫脂质体,其特征在于,还包括如下特征中的一项或几项:
a)所述磷脂选用氢化大豆卵磷脂;
b)所述环丙沙星与纳米脂质体中磷脂的摩尔比为0.1~0.5:1;
c)所述抗铜绿假单胞菌单克隆抗体与纳米脂质体中磷脂含量的摩尔比为1:100~1000。
4.如权利要求3所述的靶向铜绿假单胞菌的免疫脂质体,其特征在于,还包括以下特征中的一项或几项:
(ⅰ)所述纳米脂质体中氢化大豆卵磷脂与胆固醇的质量比为0.1~1.0:1;
(ⅱ)所述纳米脂质体中氢化大豆卵磷脂与二硬脂酰磷脂酰乙醇胺–聚乙二醇的质量比为0.1~1.0:1。
5.一种如权利要求1所述的靶向铜绿假单胞菌的免疫脂质体的制备方法,其特征在于,包括如下步骤:
步骤一、将空白脂质体与抗铜绿假单胞菌药物混合孵育,得到载药纳米脂质体;
步骤二、将抗铜绿假单胞菌单克隆抗体与连接脂质混合反应,得到单克隆抗体脂质共聚物;
步骤三、将步骤一所得载药纳米脂质体与步骤二所得单克隆抗体脂质共聚物共孵育,得到靶向铜绿假单胞菌的免疫脂质体。
6.如权利要求5所述的靶向铜绿假单胞菌的免疫脂质体的制备方法,其特征在于,还包括以下特征中的一项或几项:
(a)步骤一中所述混合孵育时间为20~40 min;
(b)步骤一中混合孵育后还需用10%蔗糖溶液透析,去除游离的抗铜绿假单胞菌药物;
(c)步骤二中所述连接脂质为DSPE-PEG2000-NHS;
(d)步骤二中所述混合反应具体为4 ℃混合过夜反应;
(e)步骤二中混合反应后还需透析去除未反应的DSPE-PEG2000-NHS;
(f)步骤三中共孵育后还需用10 mM HEPES缓冲液透析纯化,去除未插入的抗铜绿假单胞菌单克隆抗体及单克隆抗体脂质共聚物。
7.如权利要求5所述的靶向铜绿假单胞菌的免疫脂质体的制备方法,其特征在于,还包括以下特征中的一项或几项:
(1)步骤一中所述空白脂质体的制备方法包括以下步骤:
S1、先将胆固醇溶于乙醇,然后按质量配比加入磷脂和二硬脂酰磷脂酰乙醇胺–聚乙二醇,待溶解后加入硫酸铵溶液,进行水合反应,得到水合产物;
S2、将步骤S1所得的水合产物通过脂质体挤出仪制备得到脂质体混悬液;
S3、将步骤S2所得脂质体混悬液透析,得到空白脂质体;
(2)步骤二中所述抗铜绿假单胞菌单克隆抗体的制备方法包括以下步骤:
T1、构建包含所述单克隆抗体的编码基因的表达载体;
T2、通过瞬时转染宿主细胞的方法构建包含所述表达载体的宿主细胞;
T3、连续培养所述宿主细胞并收集细胞培养上清液;
T4、进行目的蛋白纯化,得到抗铜绿假单胞菌单克隆抗体。
8.如权利要求7所述的靶向铜绿假单胞菌的免疫脂质体的制备方法,其特征在于,还包括以下特征中的一项或几项:
1)特征(1)中,步骤S1中所述磷脂与胆固醇的质量比为0.1~1.0:1;
2)特征(1)中,步骤S1中所述磷脂与所述二硬脂酰磷脂酰乙醇胺–聚乙二醇的质量比为0.1~1.0:1;
3)特征(1)中,步骤S1中所述水合反应时间为20~40 min;
4)特征(1)中,步骤S3中透析所用的溶液为10%蔗糖溶液;
5)特征(2)中,步骤T1中所述表达载体为pM09表达载体;
6)特征(2)中,步骤T2中所述宿主细胞为人HEK293E细胞;
7)特征(2)中,步骤T4中所述纯化为亲和层析柱纯化。
9.如权利要求1~4任一项所述的靶向铜绿假单胞菌的免疫脂质体或权利5~8中任一项所述的制备方法制得的靶向铜绿假单胞菌的免疫脂质体在制备治疗铜绿假单胞菌感染药物和递送工具中的应用。
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