【作者】 谢志军；

【导师】 向选东；

【作者基本信息】 中南大学 ， 内科学， 2011， 硕士

【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。更多还原

【Abstract】 ObjectiveTo prepare the long circulating immunoliposomes wrapping Praziquantel against Schistosoma japonicum as the preliminary work of the targeted therapy.Methods1、Expression and purification of SIEA26-28 ku-scFvE.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 praziquantelThe 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-scFvBy 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.Results1、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%.conclusionSIEA26-28 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.更多还原