基于PSCA的肽-DNA复合疫苗的构建及其免疫学效应的检测

Construction of a PSCA based peptide-DNA combined vaccine and its CTLs killing activity identification

目的设计、制备和鉴定PLL-PSCA14-22/-pcDNA3.1(+)-PSCA复合疫苗及其免疫学效应的检测。方法将带正电荷的多聚赖氨酸(PLL)通过化学方法与PSCA的一个CTL表位肽PSCA14-22偶联,将上述阳离子多肽PLL-PSCA14-22与带负电荷的编码PSCA的重组质粒pcDNA3.1(+)-PSCA利用正负电荷相吸作用制备靶向PSCA的肽-DNA复合疫苗;通过DNA阻滞实验和DNaseⅠ保护实验鉴定制备的疫苗;将制备成功的复合疫苗体外转染Hela细胞,通过RT-PCR检测该疫苗的转染效率;并进一步通过LDH释放实验检测该疫苗对PSCA阳性的前列腺癌细胞系LNCaP的特异性杀伤效应。结果当PLL-PSCA14-22多肽与pcDNA3.1(+)-PSCA重组质粒正负电荷比≥2时,DNA电泳条带完全消失,即重组质粒pcDNA3.1(+)-PSCA完全被PLL-PSCA14-22多肽所包裹,复合疫苗可有效抵抗DNaseⅠ的消化作用。RT-PCR结果表明该复合疫苗在体外可有效转染Hela细胞,且当正负电荷比为64时出现较高的转染效率,并且能够有效激发CTL应答杀伤LNCaP细胞。结论成功构建的靶向PSCA的多肽-DNA复合疫苗可有效转染真核细胞,诱导CTL应答杀伤前列腺癌细胞,该研究为进一步体内动物实验探讨该疫苗对前列腺癌的免疫治疗效应奠定了基础。

This study is aimed to construct a PSCA based peptide-DNA combined vaccine and identify its CTLs killing activity.PLL-PSCA14-22 was obtained by coupling the positively charged poly-lysine（PLL） with a CTL epitope peptide PSCA14-22.Then DNA/peptide combined vaccine was formed by the self-assembly of a cationic peptide PLL-PSCA14-22 with a plasmid encoding human full-length PSCA gene through electrostatic interactions.The preparation of this combined vaccine was then identified with both DNA retardation and DNaseⅠ protection experiments.The transfection efficiency and CTLs killing activity were evaluated by RT-PCR and LDH release assay,respectively.The protection of DNA was begun at the charge ratios of peptide/DNA≥2,at which DNA was completely retarded in the DNA retardation assay.The PSCA transcripts was existed in the combined vaccine-transfected Hela cells and CTLs killing activity was induced in vivo,which led to the obvious killing of PSCA-positive LNCaP cells.The PSCA-based peptide-DNA combined vaccine can be transfected into eukaryotic cells and induce specific CTLs response and kill PSCA-positive LNCaP cells effectively,which may pave a way for further studies in animals.