【摘要】 目的 :探讨PEI作为免疫佐剂对G250抗原肽基因PVAX1/C-G250肽-C免疫保护效果的增强作用及联合应用CAIX蛋白疫苗进行PRIME—BOOST免疫程序免疫增强效果。方法:用Eco R I、Xho I和Eco R I、Sal I分别双酶切PVAX1及既往构建的p ET28a(+)/C-G250肽-C质粒。利用DNA重组技术构建重组质粒PVAX1/C-G250肽-C,酶切分析鉴定。大量提取质粒并分光光度计测质粒含量。将32只雌性昆明小鼠随机分为(A)裸DNA组,(B)DNA-PEI复合物组,(C)DNA-PEI+蛋白疫苗组,(D)空白对照组。按0,10,20,30天程序经股四头肌注射免疫。C组在第20天及第30天进行蛋白冲击。初次免疫前和第40天鼠尾取血,ELISA法检测抗体滴度。流式细胞术测淋巴细胞亚群CD4+和CD8+。结果:酶切及基因测序鉴定证实G250抗原肽c DNA正确插入PVAX1/C-G250肽-C真核表达的重组质粒中。昆明小鼠经4次免疫后,3个实验组都产生了特异性的体液和细胞免疫反应,B组的抗体滴度1:1.28×104及CD4+、CD8+达26.12%和12.60%,明显高于A组的1:3.2×103和CD4+,CD8+占到19.32%和10.74%。而蛋白冲击组的1:5.12×104和CD4+,CD8+占到41.96%和15.14%,明显高于B组(P>0.05)。结论:成功构建重组质粒PVAX1/C-G250肽-C。该DNA疫苗与PEI和G250蛋白疫苗联合使用后产生极强的免疫原性,诱导产生了高滴度、高特异性抗体及细胞免疫反应。证实G250DNA疫苗联合PEI使用并进行蛋白疫苗冲击的免疫策略可产生强大的免疫保护作用。为恶性肿瘤的术后辅助治疗提供新的思路和方法。更多还原

【Abstract】 Objective: Discussed PEI as an adjuvant to enhance the protective of the recombinant DNA vaccine- plasmid PVAX1/C-G250 peptide-C and a strategy of vaccination was attempted with the DNA vaccine followed by a boosts with the recombinant protein vaccine(DNA prime-protein boost vaccine). Methods: For the replacement of the enzyme digestion sites, p ET28a(+)/C-G250 peptide-C was taken as a template. C-G250 peptide-C was take from the p ET28a(+)/C-G250 peptide-C plasmid with the help of Eco R l、Sal l. The G250 peptide was then inserted to the PVAX1 which was enzyme digested by Eco R l、Xho l and obtain the recombinant plasmid PVAX1/C-G250 peptide-C. Sixteen Kunming mices was divided into fourgroups:(A)PVAX1/C-G250peptide-C DNA plasmid alone,(B)DNA-PEI polyplexes,(3)DNA prime protein boost vaccine,(4)Blank. mice were injected at 0, 10, 20, 30 days under quadriceps femoris muscle and the third group was given with the G250 peptide which was made in our prophase test at the 20 and the 30 days.Immune responses were detected by indirect ELISA and flow cytometry. Results: The recombinant plasmid PVAX1/C-G250 peptide-C was confirmed to be correct by the restriction enzyme digestion and DNA sequencing. After four times of immunization, the titer of antibody can be detected in blood serum of the first three groups of kunming mices. DNA-PEI polyplexes was higher than DNA alone.Much higher G250-specific antibody were seen in the DNA prime protein boost vaccine than DNA-PEI polyplexes and DNA alone. CD4+and CD8+ were the same trend as the titer of antibody.Conclusion: Recombinant DNA vaccine-plasmid PVAX1/C-G250 peptide-C was successfully constructed. Higher G250-specific antibody was seen by indirect ELISA. CD4+ and CD8+ lymphocytes was tested by flow cytometry. These findings demonstrated that PEI as an adjuvant was useful to enhance the protective of the DNA vaccine. DNA prime protein boost regimen can induce an enhanced antitumor effect in a heterologous prime boost protocol and suggest a new way to a successful immunization against metastatic carcinoma.更多还原