增强型Trop2-CD40L病毒样颗粒疫苗的制备、纯化及活性研究

Expression,Purification and Activity Study on the Immune-Enhanced Trop2-CD40L Virus Like Particles

目的 获得嵌合免疫增强佐剂CD40配体（CD40L）的Trop2-CD40L病毒样颗粒（VLPs）疫苗,为后续探究VLPs在肿瘤治疗中的免疫原性及免疫保护作用奠定基础。方法 通过重叠延伸PCR方法扩增以Trop2为靶点、嵌合免疫佐剂CD40L的Trop2-CD40L融合基因,构建杆状病毒表达载体pFastbacTM /Trop2-CD40L,并转化大肠埃希菌E.coli DH10bac,提取杆粒并经PCR鉴定成功命名为Trop2-CD40L Bacmid,后者转染Tn5昆虫细胞获得重组病毒Trop2-CD40L rBV,与Gag rBV共感染昆虫细胞制备重组病毒样颗粒,透析浓缩后蔗糖密度梯度离心纯化,Western Blot鉴定VLPs目的蛋白表达、电镜观察所制备VLPs形态,Cytokine ELISA方法检测Trop2-CD40L VLPs体外生物学活性。结果 成功扩增了Trop2-CD40L融合基因,通过杆状病毒表达系统、借助SIV Gag衣壳蛋白装配特点成功制备了增强型Trop2-CD40L 病毒样颗粒,与Trop2 VLPs比较具有更好的生物学活性,差异具有统计学意义（t=7.266,,P〈0.05）。结论 制备出具有一定生物学活性的增强型Trop2-CD40L病毒样颗粒,证实了CD40L作为免疫佐剂使用有免疫增强效应。

Objective To construct the immune-enhanced Virus like Particles （VLPs） based on Trop2-CD40L fusion protein, which laid the foundation for the subsequent induction of humoral and cellular immune response. Methods Firstly,amplified a Trop2-CD40L fusion gene by the method of over-lapping PCR, then cloned the fusion gene into pFastbacTM shuttle vector （named as pFastbac^TM/Trop2-CD40L）. The constructed vector was then transformed into E. coli DH10Bac to build recombi- nant Trop2-CD40L Bacmid. The rBV/Trop2-CD40L was obtained by transfection TN5 insect cells with the Trop2-CD40L Bacmid,and finally,the Trop2-CD40L VLPs was prepared by co-infection TN5 cells with rBV/Gag and rBV/Trop2-CD40L in a MOI ratio of 1 ： 10. After large scale preparation, VLPs was concentrated and purified bysucrose density gradient cen- trifugation. Then WB detection, electron microscope and Cytokine ELISA were performed to confirm the Trop2-CD40L VLPs production and it＇s in vitro biological activity（t= 7. 266 ,P〈0.05）. Results Successfully amplified the Trop2-CD40L fusion gene by the method of overlapping PCR and expressed and purified Trop2-CD40L chimeric VLPs by using the Bac-to- Bac baculovirus expression system （SIV Gag was used as shell protein）. Conclusion The Trop2-CD40L chimeric VLPs was successfully prepared, evidencing CD40L used as immune adjuvant immune activator,which established a fine theory and experiment basis for the further research.