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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。
Objective To design, prepare and identify the PLL-PSCA14-22 / -pcDNA3.1 (+) - PSCA composite vaccine and its immunological effects. Methods The positively charged polylysine (PLL) was chemically coupled to a CTL epitope peptide PSCA14-22 of PSCA. The cationic peptide PLL-PSCA14-22 and negatively charged recombinant plasmid encoding PSCA pcDNA3.1 (+) - PSCA was used to prepare PSCA-targeting peptide-DNA composite vaccine by positive and negative phase adsorption. DNA vaccine and DNaseⅠprotection were used to identify the vaccine. The recombinant vaccine was transfected into Hela The transfection efficiency of the vaccine was tested by RT-PCR. The specific killing effect of the vaccine on LNCaP, a PSCA-positive prostate cancer cell line, was further tested by LDH release assay. Results When the positive and negative charge ratio of PLL-PSCA14-22 polypeptide to pcDNA3.1 (+) - PSCA recombinant plasmid was more than or equal to 2, the electrophoresis band of DNA disappeared completely, that is, the recombinant plasmid pcDNA3.1 (+) - PSCA was completely blocked by PLL-PSCA14 -22 polypeptide package, the composite vaccine can effectively resist DNase Ⅰ digestion. The results of RT-PCR indicated that the recombinant vaccine could efficiently transfect Hela cells in vitro and showed high transfection efficiency when the ratio of positive and negative charges was 64, and could effectively stimulate the CTL response to kill LNCaP cells. CONCLUSION: The PSCA-targeting peptide-DNA composite vaccine successfully constructed can effectively transfect eukaryotic cells and induce CTL responses to kill prostate cancer cells. This study laid the foundation for further in vivo animal experiments to explore the immunotherapeutic effect of the vaccine against prostate cancer.