CN114767876A - 一种搭载噬菌体的复合多孔微球及其制备方法 - Google Patents
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Abstract
本发明公开了一种搭载噬菌体的复合多孔微球及其制备方法。复合多孔微球是由丝素微球搭载结合噬菌体形成;通过支链聚乙烯亚胺修饰微球表面电位转为正电位,通过静电引力吸附负电位的噬菌体,形成搭载噬菌体的复合多孔微球。本发明的复合多孔微球可搭载不同功能化的噬菌体从而呈现出不同的功能,并且复合多孔微球可以通过微创手术,使用注射器直接注射到病灶位置,为脑梗死等诸多疾病的治疗提供了新的方法和材料。
Description
技术领域
本发明属于生物医用材料领域的一种复合微球及其制备方法,尤其是涉及了一种用于脑梗死修复的搭载噬菌体的复合多孔微球及其制备方法。
背景技术
脑组织是人体的中枢神经系统,主导人体体内的一切活动过程。脑梗死可以造成人体残疾,引发肢体活动障碍、偏瘫、意识障碍和痴呆等,目前没有有效的医学手段可以直接修复受损的脑组织。目前,脑梗死在急性期后的治疗方法是高压氧治疗、康复训练、服用神经营养因子或抗炎药物,但是这些治疗方法会造成一些并发症,例如氧中毒和气压伤等,而且治疗效果非常有限。在梗死后,受损脑组织中新生的血管网络和神经网络对于病人的后期恢复是非常重要的。但是对于目前来说,同时在脑梗死区促进血管生成和神经生成依然是一个巨大的挑战。
现有的丝素蛋白微球采用乳液法和冻干法制备,功能单一,虽然生物相容性良好但是不能刺激神经干细胞向神经元分化并且不能刺激血管生成。
发明内容
针对现有生物材料对脑梗死治疗效果不理想等主要问题,本发明提供了一种以丝素蛋白为主要支架,以噬菌体为功能因子,采用乳液法、冷冻干燥以及静电吸附制备的搭载噬菌体的复合多孔微球,结合了噬菌体和丝素蛋白。
本发明制备的搭载噬菌体的复合多孔微球可以用于脑梗死的修复,可有效促进脑梗死部位血管再生和神经再生等问题,增强动物肢体功能恢复。本发明很好地利用了脑梗死区域形成的空穴空间,实现了脑梗死的修复。
而且,噬菌体为纳米纤维,没有高级三维结构,无法搭载神经干细胞,本发明通过特殊的制备也解决了噬菌体难以结合于神经干细胞的技术问题。
本发明的技术方案是:
一、一种搭载噬菌体的复合多孔微球:
所述的复合多孔微球是由丝素微球搭载结合噬菌体形成。所述的复合多孔微球的组成成分是丝素和噬菌体。
所述的噬菌体包含裂解性噬菌体、温和型噬菌体、野生型噬菌体以及基因工程化噬菌体等噬菌体。
所述的多孔微球的孔隙率在80%以上,大小约为150–250μm,噬菌体搭载量为7.45×102PFU/mg-6.51×1010PFU/mg。
所述的复合多孔微球在治疗大脑梗死和制备治疗大脑梗死药物中的应用。
二、一种搭载噬菌体的复合多孔微球的制备方法:
S1、由蚕茧制备获得多孔丝素微球;
S2、对多孔丝素微球进行去菌处理;
S3、在多孔丝素微球上复合搭载制备噬菌体形成复合多孔微球。
所述的步骤S1具体为:
S11、将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2w/v%的丝素蛋白溶液;
S12、将石油醚在-80℃冷冻1h;
S13、取S12冷冻获得的石油醚于烧杯中,加入司盘80(span-80),使混合均匀;
S14、将S11获得的丝素蛋白溶液加入S13获得的石油醚溶液,搅拌;
S15、将S14搅拌获得的溶液迅速倒入S12冷冻获得的石油醚中,再置于-20℃冰箱静置沉降,在-20℃氛围下除去溶液中的石油醚,获得含冰晶的丝素蛋白微球沉淀,将丝素蛋白微球沉淀进行冷冻真空干燥即可制备出多孔丝素微球。
所述的步骤S2具体为:
S21、将多孔丝素微球浸入无水乙醇中静置48h;
S22、将无水乙醇处理后的多孔丝素微球转移至体积分数为75%的乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
S23、将乙醇溶液中的多孔丝素微球用无菌PBS缓冲液洗涤10次,去除乙醇,将多孔丝素微球分散在PBS缓冲液中保存。
所述的步骤S3具体为:
S31、将无菌支链聚乙烯亚胺溶液加入到多孔丝素微球中孵育,获得了吸附支链聚乙烯亚胺的多孔丝素微球;
S32、将吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS缓冲液中,用PBS缓冲液洗涤10次,去除游离的支链聚乙烯亚胺成分;
S33、扩增并纯化噬菌体,制备噬菌体溶液;
S34、将噬菌体溶液加入到S32获得的吸附支链聚乙烯亚胺的多孔丝素微球溶液中,震荡孵育形成搭载噬菌体的复合多孔微球;
S35、将S34获得的搭载噬菌体的复合多孔微球用无菌PBS缓冲液洗涤5次,去除游离的噬菌体,得到纯化的搭载噬菌体的复合多孔微球。
本发明在丝素微球搭载噬菌体复合制备过程中加入了支链聚乙烯亚胺,利用丝素微球和噬菌体的电荷差异加入支链聚乙烯亚胺以静电吸附方式成功将丝素微球搭载噬菌体复合,制备出与复合丝素微球表面噬菌体电负性相同的负电荷的多孔丝素微球,实现了噬菌体的饱和吸附,使丝素微球表面被噬菌体高度功能化,对后期体内治疗脑梗死带来更佳的修复效果。
本发明中的丝素微球通过支链聚乙烯亚胺处理改变其表面电位,由负电位转换为正电位,能够使噬菌体通过静电引力稳定吸附在丝素微球表面。
本发明所述的搭载噬菌体的复合多孔微球表面上搭载的噬菌体可以增加神经干细胞对丝素微球的吸附,使微球表面搭载更多神经干细胞。
搭载神经干细胞后,所述的复合多孔微球通过注射器直接注射进入大脑梗死部位,在大脑梗死部位强烈诱导血管生成和神经生成。
所述的复合多孔微球载入到神经干细胞中,由神经干细胞注射进入大脑梗死部位,进行针对性处理。
所述多孔微球以丝素蛋白为基质材料,形成孔隙率在80%以上,大小约为150–250μm,表面电位为负电位的多孔微球,通过支链聚乙烯亚胺修饰微球表面电位转为正电位,通过静电引力吸附负电位的噬菌体,形成搭载噬菌体的复合多孔微球。
本发明所述的复合多孔微球可以搭载不同功能化的噬菌体从而呈现出不同的功能,并且复合多孔微球可以通过微创手术,使用注射器直接注射到病灶位置,为脑梗死等诸多疾病的治疗提供了新的方法和材料。
与现有技术相比,本发明具有以下突出特点:
1)优良的生物相容性:丝素蛋白作为一种天然蛋白分子,对人体不会产生毒性,不会引起人体免疫反应;噬菌体是存在于人体的细菌病毒,不会对人体产生毒性;
2)可注射性:搭载噬菌体的复合多孔微球可以通过微创手术,使用注射器直接注射于脑梗死部位,为病人减轻手术痛苦;
3)促进脑组织修复:搭载噬菌体的复合多孔微球可以搭载神经干细胞,在脑梗死部位诱导神经干细胞分化为神经元和胶质细胞;噬菌体可以强烈诱导血管网络的生成,为神经再生提供氧气和营养物质;丝素微球为细胞浸润,增殖和分化提供物理支撑;
4)多功能性:噬菌体可以通过基因工程将功能性的多肽展示在噬菌体表面形成功能化的噬菌体,搭载不同功能化的噬菌体可以使复合多孔微球具备多种不同的功能;
5)成本低廉,环保无污染。
附图说明
图1为实施例2中微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果免疫荧光图。
图2为实施例3中微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果免疫荧光图。
图3为实施例4中微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果免疫荧光图。
图4为实施例5中微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果免疫荧光图。
图5为实施例2,3,4,5中微球在第12周对脑梗死部位神经生成的促进效果免疫荧光图。
具体实施方式
下面结合实施例对本发明作进一步的阐述,以下实施例仅为本发明的优选实施例,并不限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化,凡在本发明的精神和原则之内,所作的的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
本发明的实施例具体如下:
实施例1
1)将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2%(w/v)的丝素蛋白溶液;
2)取500mL石油醚在-80℃冷冻1h;
3)取常温30mL石油醚,加入0.7mL span-80,使两者混合均匀;
4)将5mL的1)中的丝素蛋白溶液加入3)中的石油醚溶液,600rpm搅拌20min;
5)将4)溶液迅速倒入2)中冷冻的石油醚中,再置于-20℃冰箱静置沉降;
6)在-20℃氛围下除去5)溶液中的石油醚,获得含冰晶的丝素微球沉淀,将该丝素微球沉淀进行冷冻真空干燥可获得干燥的多孔丝素微球;
7)将6)中干燥的多孔丝素蛋白微球浸入无水乙醇中静置48h;
8)将7)无水乙醇中的多孔丝素微球转移至75%乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
9)将8)75%乙醇溶液中的多孔丝素微球用无菌PBS洗涤10次,去除乙醇,即得到多孔丝素微球。
所得的多孔丝素微球的组成成分是丝素,所述的多孔微球的孔隙率在80%以上,大小约为150-250μm,噬菌体含量为0PFU/mg,表面电位为-24mV。
实施例2
1)将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2%(w/v)的丝素蛋白溶液;
2)取500mL石油醚在-80℃冷冻1h;
3)取常温30mL石油醚,加入0.7mL span-80,使两者混合均匀;
4)将5mL的1)中的丝素蛋白溶液加入3)中的石油醚溶液,600rpm搅拌20min;
5)将4)溶液迅速倒入2)中冷冻的石油醚中,再置于-20℃冰箱静置沉降;
6)在-20℃氛围下除去5)溶液中的石油醚,获得含冰晶的丝素微球沉淀,将该丝素微球沉淀进行冷冻真空干燥可获得干燥的多孔丝素微球;
7)将6)中干燥的多孔丝素蛋白微球浸入无水乙醇中静置48h;
8)将7)无水乙醇中的多孔丝素微球转移至75%乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
9)将8)75%乙醇溶液中的多孔丝素微球用无菌PBS洗涤10次,去除乙醇,将多孔丝素微球分散在PBS中保存;
10)将5%的无菌支链聚乙烯亚胺溶液加入到9)PBS中的多孔丝素微球中,50rpm震荡孵育24h;
11)将10)中的吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS中,用PBS洗涤10次,去除游离的支链聚乙烯亚胺成分,即得到经过支链聚乙烯亚胺修饰的多孔丝素微球。
所得的搭载噬菌体的复合多孔微球的组成成分是丝素和支链聚乙烯亚胺,所述的多孔微球孔隙率在80%以上,大小约为150-250μm,噬菌体含量为0PFU/mg,表面电位为+9.26mV。
本实施例的微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果如图1所示。
实施例3
1)将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2%(w/v)的丝素蛋白溶液;
2)取500mL石油醚在-80℃冷冻1h;
3)取常温30mL石油醚,加入0.7mL span-80,使两者混合均匀;
4)将5mL的1)中的丝素蛋白溶液加入3)中的石油醚溶液,600rpm搅拌20min;
5)将4)溶液迅速倒入2)中冷冻的石油醚中,再置于-20℃冰箱静置沉降;
6)在-20℃氛围下除去5)溶液中的石油醚,获得含冰晶的丝素微球沉淀,将该丝素微球沉淀进行冷冻真空干燥可获得干燥的多孔丝素微球;
7)将6)中干燥的多孔丝素蛋白微球浸入无水乙醇中静置48h;
8)将7)无水乙醇中的多孔丝素微球转移至75%乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
9)将8)75%乙醇溶液中的多孔丝素微球用无菌PBS洗涤10次,去除乙醇,将多孔丝素微球分散在PBS中保存;
10)将5%的无菌支链聚乙烯亚胺溶液加入到9)中的多孔丝素微球中,50rpm震荡孵育24h;
11)将10)中的吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS中,用PBS洗涤10次,去除游离的支链聚乙烯亚胺成分;
12)将噬菌体加入1mL的E.coil.ER2738菌液中。37℃静置孵育60min后,将菌液置于37℃摇床中,220rpm培养1h。随后将菌液倒入1L的LB培养基中,220rpm培养24h。将菌液8000×g离心20min,将上清液转入新的烧杯中,加入30g/L NaCl and 40g/L PEG-8000,4℃沉降8h。8000×g,4℃离心50min,去掉上清。加入10mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。8000×g,4℃离心30min去除杂质。向上清中加入0.15倍体积的PEG/NaCl溶液沉降噬菌体。8000×g,4℃离心30min,去掉上清。加入1mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。13000×g,4℃离心20min去除杂质获得噬菌体溶液。
13)将12)中的浓度为1×105PFU/mL的噬菌体溶液加入到11)中的吸附支链聚乙烯亚胺的多孔丝素微球溶液中,50rpm震荡孵育24h,形成搭载噬菌体的复合多孔微球;
14)将13)中的搭载噬菌体的复合多孔微球用无菌PBS洗涤5次,去除游离的噬菌体,即得到纯化的搭载噬菌体的复合多孔微球。
所得的搭载噬菌体的复合多孔微球的组成成分是丝素和支链聚乙烯亚胺,所述的多孔微球孔隙率在80%以上,大小约为150-250μm,噬菌体含量为7.45×102PFU/mg,表面电位为+8.48mV。
本实施例的微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果如图2所示。
实施例4
1)将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2%(w/v)的丝素蛋白溶液;
2)取500mL石油醚在-80℃冷冻1h;
3)取常温30mL石油醚,加入0.7mL span-80,使两者混合均匀;
4)将5mL的1)中的丝素蛋白溶液加入3)中的石油醚溶液,600rpm搅拌20min;
5)将4)溶液迅速倒入2)中冷冻的石油醚中,再置于-20℃冰箱静置沉降;
6)在-20℃氛围下除去5)溶液中的石油醚,获得含冰晶的丝素微球沉淀,将该丝素微球沉淀进行冷冻真空干燥可获得干燥的多孔丝素微球;
7)将6)中干燥的多孔丝素蛋白微球浸入无水乙醇中静置48h;
8)将7)无水乙醇中的多孔丝素微球转移至75%乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
9)将8)75%乙醇溶液中的多孔丝素微球用无菌PBS洗涤10次,去除乙醇,将多孔丝素微球分散在PBS中保存;
10)将5%的无菌支链聚乙烯亚胺溶液加入到9)中的多孔丝素微球中,50rpm震荡孵育24h;
11)将10)中的吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS中,用PBS洗涤10次,去除游离的支链聚乙烯亚胺成分;
12)将噬菌体加入1mL的E.coil.ER2738菌液中。37℃静置孵育60min后,将菌液置于37℃摇床中,220rpm培养1h。随后将菌液倒入1L的LB培养基中,220rpm培养24h。将菌液8000×g离心20min,将上清液转入新的烧杯中,加入30g/L NaCl and 40g/L PEG-8000,4℃沉降8h。8000×g,4℃离心50min,去掉上清。加入10mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。8000×g,4℃离心30min去除杂质。向上清中加入0.15倍体积的PEG/NaCl溶液沉降噬菌体。8000×g,4℃离心30min,去掉上清。加入1mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。13000×g,4℃离心20min去除杂质获得噬菌体溶液。
13)将12)中的浓度为1×109PFU/mL的噬菌体溶液加入到11)中的吸附支链聚乙烯亚胺的多孔丝素微球溶液中,50rpm震荡孵育24h,形成搭载噬菌体的复合多孔微球;
14)将13)中的搭载噬菌体的复合多孔微球用无菌PBS洗涤5次,去除游离的噬菌体,即得到纯化的搭载噬菌体的复合多孔微球。
所得的搭载噬菌体的复合多孔微球的组成成分是丝素和支链聚乙烯亚胺,所述的多孔微球孔隙率在80%以上,大小约为150-250μm,噬菌体含量为1.96×107PFU/mg,表面电位为+5.01mV。
本实施例的微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果如图3所示。
实施例5
1)将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2%(w/v)的丝素蛋白溶液;
2)取500mL石油醚在-80℃冷冻1h;
3)取常温30mL石油醚,加入0.7mL span-80,使两者混合均匀;
4)将5mL的1)中的丝素蛋白溶液加入3)中的石油醚溶液,600rpm搅拌20min;
5)将4)溶液迅速倒入2)中冷冻的石油醚中,再置于-20℃冰箱静置沉降;
6)在-20℃氛围下除去5)溶液中的石油醚,获得含冰晶的丝素微球沉淀,将该丝素微球沉淀进行冷冻真空干燥可获得干燥的多孔丝素微球;
7)将6)中干燥的多孔丝素蛋白微球浸入无水乙醇中静置48h;
8)将7)无水乙醇中的多孔丝素微球转移至75%乙醇溶液中静置1h,杀灭多孔丝素微球中的细菌;
9)将8)75%乙醇溶液中的多孔丝素微球用无菌PBS洗涤10次,去除乙醇,将多孔丝素微球分散在PBS中保存;
10)将5%的无菌支链聚乙烯亚胺溶液加入到9)中的多孔丝素微球中,50rpm震荡孵育24h;
11)将10)中的吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS中,用PBS洗涤10次,去除游离的支链聚乙烯亚胺成分;
12)将噬菌体加入1mL的E.coil.ER2738菌液中。37℃静置孵育60min后,将菌液置于37℃摇床中,220rpm培养1h。随后将菌液倒入1L的LB培养基中,220rpm培养24h。将菌液8000×g离心20min,将上清液转入新的烧杯中,加入30g/L NaCl and 40g/L PEG-8000,4℃沉降8h。8000×g,4℃离心50min,去掉上清。加入10mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。8000×g,4℃离心30min去除杂质。向上清中加入0.15倍体积的PEG/NaCl溶液沉降噬菌体。8000×g,4℃离心30min,去掉上清。加入1mL的PBS将沉淀重新悬浮,37℃,220rpm震荡1h。13000×g,4℃离心20min去除杂质获得噬菌体溶液。
13)将12)中的浓度为1×1013PFU/mL的噬菌体溶液加入到11)中的吸附支链聚乙烯亚胺的多孔丝素微球溶液中,50rpm震荡孵育24h,形成搭载噬菌体的复合多孔微球;
14)将13)中的搭载噬菌体的复合多孔微球用无菌PBS洗涤5次,去除游离的噬菌体,即得到纯化的搭载噬菌体的复合多孔微球。
所得的搭载噬菌体的复合多孔微球的组成成分是丝素和支链聚乙烯亚胺,所述的多孔微球大小约为150-250μm,噬菌体含量为6.51×1010PFU/mg,表面电位为-5.9mV。
本实施例的微球在第2周对脑梗死部位血管生成(a)和神经生成(b)的促进效果如图4所示。
最终如图5所示,根据本发明研究发现,将搭载噬菌体和神经干细胞的复合丝素微球注射到脑梗死部位后,在植入后第2周,相比于没有搭载噬菌体的实施例2,搭载不同噬菌体含量的实施例3-5能够在2周内明显刺激脑梗死部位的血管生成并且神经干细胞能够向神经元分化。并且噬菌体搭载量最高的实施例5的血管生成和神经元分化的效果最强。在第12周,相比于实施例2,实施例3-5微球周围出现明显的神经纤维,并且实施例4和实施例5微球植入部位生成出大量致密的新生的神经组织。结果表明搭载噬菌体的复合多孔微球能够显著刺激脑梗死部位生成血管和神经组织。
Claims (8)
1.一种搭载噬菌体的复合多孔微球,其特征在于:
所述的复合多孔微球是由丝素微球搭载结合噬菌体形成。
2.根据权利要求1所述的一种搭载噬菌体的复合多孔微球,其特征在于:
所述的噬菌体包含裂解性噬菌体、温和型噬菌体、野生型噬菌体以及基因工程化噬菌体等噬菌体。
3.根据权利要求1所述的一种搭载噬菌体的复合多孔微球,其特征在于:
所述的多孔微球的孔隙率在80%以上,大小约为150–250μm,噬菌体搭载量为7.45×102PFU/mg-6.51×1010PFU/mg。
4.权利要求1所述复合多孔微球的应用,其特征在于:
所述的复合多孔微球在治疗大脑梗死和制备治疗大脑梗死药物中的应用。
5.权利要求1-4任一所述复合多孔微球的制备方法,其特征在于:
S1、由蚕茧制备获得多孔丝素微球;
S2、对多孔丝素微球进行去菌处理;
S3、在多孔丝素微球上复合搭载制备噬菌体形成复合多孔微球。
6.根据权利要求5所述的复合多孔微球的制备方法,其特征在于:
所述的步骤S1具体为:
S11、将蚕茧茧壳剪碎、脱胶、洗涤、干燥、溶解、透析和除杂后制备成2w/v%的丝素蛋白溶液;
S12、将石油醚在-80℃冷冻1h;
S13、取S12冷冻获得的石油醚于烧杯中,加入司盘80,使混合均匀;
S14、将S11获得的丝素蛋白溶液加入S13获得的石油醚溶液,搅拌;
S15、将S14搅拌获得的溶液【迅速】倒入S12冷冻获得的石油醚中,再置于-20℃冰箱静置沉降,在-20℃氛围下除去溶液中的石油醚,获得含冰晶的丝素蛋白微球沉淀,将丝素蛋白微球沉淀进行冷冻真空干燥即可制备出多孔丝素微球。
7.根据权利要求5所述的复合多孔微球的制备方法,其特征在于:
所述的步骤S2具体为:
S21、将多孔丝素微球浸入无水乙醇中静置48h;
S22、将无水乙醇处理后的多孔丝素微球转移至体积分数为75%的乙醇溶液中静置,杀灭多孔丝素微球中的细菌;
S23、将乙醇溶液中的多孔丝素微球用无菌PBS缓冲液洗涤,去除乙醇,将多孔丝素微球分散在PBS缓冲液中保存。
8.根据权利要求5所述的复合多孔微球的制备方法,其特征在于:
所述的步骤S3具体为:
S31、将无菌支链聚乙烯亚胺溶液加入到多孔丝素微球中孵育,获得了吸附支链聚乙烯亚胺的多孔丝素微球;
S32、将吸附支链聚乙烯亚胺的多孔丝素微球转移至无菌PBS缓冲液中,用PBS缓冲液洗涤,去除游离的支链聚乙烯亚胺成分;
S33、扩增并纯化噬菌体,制备噬菌体溶液;
S34、将噬菌体溶液加入到S32获得的吸附支链聚乙烯亚胺的多孔丝素微球溶液中,震荡孵育形成搭载噬菌体的复合多孔微球;
S35、将S34获得的搭载噬菌体的复合多孔微球用无菌PBS缓冲液洗涤,去除游离的噬菌体,得到纯化的搭载噬菌体的复合多孔微球。
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