鼠源性HSP70、GM-CSF双修饰前列腺癌全细胞疫苗的建立与鉴定

摘要

目的：建立鼠源性HSP70、GM-CSF双修饰前列腺癌全细胞疫苗。方法通过化学合成和PCR方法分别构建小鼠Hspa1a、Csf2基因片段，TA克隆入pMD-18T载体测序验证。上述合成好的目的序列通过限制性内切酶BamHI/AscI酶切、T4DNA连接酶连接，分别装入慢病毒表达载体pLenti6.3_MCS中，构建慢病毒重组载体pLen-ti6.3_MCS_Hspa1a_cMyc 和 pLenti6.3_MCS_Csf2_V5。载有重组载体 pLenti6.3_MCS_Hspa1a_cMyc 和 pLen-ti6.3_MCS_Csf2_V5的慢病毒液分别对鼠前列腺癌细胞系RM-1进行稳定转染或共转染，Western blot检测蛋白表达，10 Gy 放射线照射使其丧失增殖能力后冻存。结果测序验证重组慢病毒表达载体 pLen-ti6.3_MCS_Hspa1a_cMyc和pLenti6.3_MCS_Csf2_V5构建成功。经293T细胞包装后分别或共转染RM-1，Western blot证实GM-CSF、sHSP70表达，GM-CSF和HSP70共表达。结论本实验成功建立了鼠HSP70、GM-CSF双修饰RM-1细胞全细胞疫苗，为以后进一步探讨全细胞抗原作用机制和全细胞免疫增强机理奠定基础。%Objective To construct a whole-cell vaccine modified by mice HSP70 and GM-CSF in prostate cancer. Methods Sequence fragments carrying mice Hspa1a coupled with C-myc or Csf2 coupled with V5 tag were constructed by chemical synthesis and PCR amplification , then both fragments were cloned into the vector pMD-18T. After se quencing, fragments described above were cloned into pLenti6.3_MCS lentiviral vector. The lentiviral expression vectors were named as pLenti6.3_MCS_Hspa1a_cMyc and pLenti6.3_MCS_Csf2_V5. These vectors were transfected or co-transfected into mice prostate cancer cell line RM-1 and proteins were detected by Western blotting. Following irradia-tion of 10 Gy, cells lost proliferative ability and were frozen and then stored. Results Recombinant lentiviral vectors pLenti6.3_MCS_Hspa1a_cMyc and pLenti6.3_MCS_Csf2_V5 were successfully constructed and GM-CSF, HSP70 or both expression in the transfected RM-1 cells were verified by Western blotting. Conlusion The RM-1 whole-cell vaccines modified by HSP70 and GM-CSF have been successfully constructed, which facilitate further investigation of the action mechanisms of whole-cell antigen and immune enhancement.