抗日本血吸虫SIEA26-28KU-scFv-吡喹酮长循环免疫脂质体的制备
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摘要
目的
制备针对日本血吸虫的第三代吡喹酮长循环免疫脂质体,为日本血吸虫病的靶向性治疗奠定工作基础。
方法
1、大量表达并纯化SIEA26-28 ku-scFv
大量培养带有pET32a/SIEA26-28 ku-scFv质粒的BL21(DE3)大肠杆菌,IPTG诱导SIEA26-28 ku-scFv表达,用Ni2+柱纯化目的蛋白,将纯化蛋白进行蛋白浓度测定、SDS-PAGE检测、Western Blot鉴定。
2、制备吡喹酮长循环脂质体
采用薄膜蒸发超声分散法制备吡喹酮长循环脂质体并测定其包封率。
3、制备携带单链抗体的长循环免疫脂质体
采用新型异型双功能交联剂SPDP交联抗体与脂质体,制备第三代包裹吡喹酮的长循环免疫脂质体,计算抗体偶联率。
结果
1、大量表达SIEA26-28 ku-scFv,通过Ni2+螯合亲和层析法提纯单链抗体,通过Western Blot检测证明单链抗体能特异性识别日本血吸虫SIEA。
2、成功制备吡喹酮长循环脂质体,计算其包封率为56.1%。
3、成功制备携带单链抗体与吡喹酮的第三代长循环免疫脂质体,计算抗体偶连率为21.3%。
结论
成功提取和纯化单链抗体,制备携带单链抗体和吡喹酮的第三代长循环免疫脂质体,为下一步行靶向治疗血吸虫病奠定了工作基础。
Objective
To prepare the long circulating immunoliposomes wrapping Praziquantel against Schistosoma japonicum as the preliminary work of the targeted therapy.
Methods
1、Expression and purification of SIEA26-28 ku-scFv
E.coli BL21 (DE3) containing plasmid pET32a/SIEA26-28 ku-scFv was induced by IPTG. The expressed proteins were purified by Ni2+ column affinity chromatography. The concentration of purified proteins was determined. The purified proteins were identified by SDS-PAGE and Western blot.
2、Preparation of the long circulating liposomes wrapping praziquantel
The long circulating liposomes wrapping praziquantel were prepared by the method of thin film evaporation and Ultrasonic dispersion.And its envelopment efficiency was determined.
3、Preparation of the long circulating immunoliposomes with SIEA26-28KU-scFv
By the new shaped bifunctional crosslinker SPDP, SIEA26-28 ku-scFv was connected to liposomes in order to construct the third generation of long circulating immunoliposomes wrapping praziquantel. And its coupling rate was calculated.
Results
1、SIEA26-28 ku-scFv was highly expressed.The proteins were purified by Ni2+column affinity chromatography. Western blot showed that SIEA26-28 scFv can specifically recognize SIEA.
2、The Long circulating liposomes wrapping praziquantel were prepared successfully.And its envelopment efficiency was 56.1%.
3、The third generation of long circulating immunoliposomes wrapping praziquantel that coupled with SIEA26-28 ku-scFv were constructed successfully. And its coupling rate was 21.3%.
conclusion
SIEA26-28 ku-scFv was extracted and purified successfully. The third generation of long circulating immunoliposomes with SIEA26-28 ku-scFv and Praziquantel were prepared,which laid the basic work of the targeted therapy against Schistosoma japonicum for the next step.
制备针对日本血吸虫的第三代吡喹酮长循环免疫脂质体,为日本血吸虫病的靶向性治疗奠定工作基础。
方法
1、大量表达并纯化SIEA26-28 ku-scFv
大量培养带有pET32a/SIEA26-28 ku-scFv质粒的BL21(DE3)大肠杆菌,IPTG诱导SIEA26-28 ku-scFv表达,用Ni2+柱纯化目的蛋白,将纯化蛋白进行蛋白浓度测定、SDS-PAGE检测、Western Blot鉴定。
2、制备吡喹酮长循环脂质体
采用薄膜蒸发超声分散法制备吡喹酮长循环脂质体并测定其包封率。
3、制备携带单链抗体的长循环免疫脂质体
采用新型异型双功能交联剂SPDP交联抗体与脂质体,制备第三代包裹吡喹酮的长循环免疫脂质体,计算抗体偶联率。
结果
1、大量表达SIEA26-28 ku-scFv,通过Ni2+螯合亲和层析法提纯单链抗体,通过Western Blot检测证明单链抗体能特异性识别日本血吸虫SIEA。
2、成功制备吡喹酮长循环脂质体,计算其包封率为56.1%。
3、成功制备携带单链抗体与吡喹酮的第三代长循环免疫脂质体,计算抗体偶连率为21.3%。
结论
成功提取和纯化单链抗体,制备携带单链抗体和吡喹酮的第三代长循环免疫脂质体,为下一步行靶向治疗血吸虫病奠定了工作基础。
Objective
To prepare the long circulating immunoliposomes wrapping Praziquantel against Schistosoma japonicum as the preliminary work of the targeted therapy.
Methods
1、Expression and purification of SIEA26-28 ku-scFv
E.coli BL21 (DE3) containing plasmid pET32a/SIEA26-28 ku-scFv was induced by IPTG. The expressed proteins were purified by Ni2+ column affinity chromatography. The concentration of purified proteins was determined. The purified proteins were identified by SDS-PAGE and Western blot.
2、Preparation of the long circulating liposomes wrapping praziquantel
The long circulating liposomes wrapping praziquantel were prepared by the method of thin film evaporation and Ultrasonic dispersion.And its envelopment efficiency was determined.
3、Preparation of the long circulating immunoliposomes with SIEA26-28KU-scFv
By the new shaped bifunctional crosslinker SPDP, SIEA26-28 ku-scFv was connected to liposomes in order to construct the third generation of long circulating immunoliposomes wrapping praziquantel. And its coupling rate was calculated.
Results
1、SIEA26-28 ku-scFv was highly expressed.The proteins were purified by Ni2+column affinity chromatography. Western blot showed that SIEA26-28 scFv can specifically recognize SIEA.
2、The Long circulating liposomes wrapping praziquantel were prepared successfully.And its envelopment efficiency was 56.1%.
3、The third generation of long circulating immunoliposomes wrapping praziquantel that coupled with SIEA26-28 ku-scFv were constructed successfully. And its coupling rate was 21.3%.
conclusion
SIEA26-28 ku-scFv was extracted and purified successfully. The third generation of long circulating immunoliposomes with SIEA26-28 ku-scFv and Praziquantel were prepared,which laid the basic work of the targeted therapy against Schistosoma japonicum for the next step.
引文
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[3]张冉,易新元,曾宪芳.血吸虫病DNA疫苗的研究进展[J].中国热带医学,2004,4(1)：128-130.
[4]曹建平,李小红,刘述先.我国血吸虫疫苗研究进展和展望[J].中华传染病杂志,2005,3(6)：418-420.
[5]Fenwick A,Webster JP. Schistosomiasis:challenges for control,treatment anddrug resistance [J]. Curr Opin Infect Dis,2006,19 (6):577-582.
[6]Bergquist R, Al-Sherbiny M, Barakat R, et al. Blueprint for schistosomiasis vaccine development[J]. Acta Trop,2002,82(2):183-192.
[7]江艳,蒋作君.血吸虫药物研究概况[J].中国血吸虫病防治杂志,2001,13(1)：59-60.
[8]UNDP/World Bank/WHO/TDR.Meeting on strategies for the development of a schistosomiasis vaccine[J]. TDR/SCH/VAC-DEV,1991,3:1-11.
[9]汪世平,赵慰先,周汩波,等.日本血吸虫卵的体外培养与抗卵胚发育研究[J].中华医学杂志,1996,76(3)：218-221.
[10]汪世平,周泪波,沈国励,等.日本血吸虫未成熟卵26/28kDa抗原的诱导抗雌虫生殖和抗虫卵胚胎发育免疫的研究[J].中国寄生虫学与寄生虫病杂志1997,15(2)：79-83.
[11]Andrew RM,Bradbury,James D.Antibodies from phage antibody libraries[J]. Journal of Immunological Methods,2004,290:29-49.
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