基于SOE-PCR的核酸疫苗pIRES2-MLAA34-HSP70的构建及免疫效果研究
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摘要
目的构建pIRES2-MLAA34-HSP70重组质粒,并检测其免疫效果。方法采用逆转录PCR(RT-PCR)的方法提取急性单核细胞白血病相关抗原基因MLAA-34和热休克蛋白(HSP)70基因,设计特异性重叠引物,以重叠延伸PCR(SOEPCR)技术扩增MLAA34-HSP70融合基因,构建核酸疫苗pIRES2-MLAA34-HSP70。将核酸疫苗免疫BALB/c小鼠,检测小鼠脾淋巴细胞对U937细胞的杀伤作用及小鼠脾细胞悬液中白细胞介素(IL)-2、IL-4和γ干扰素(IFN-γ)水平。结果扩增出MLAA34-HSP70融合基因2 956bp,成功构建了核酸疫苗pIRES2-MLAA34-HSP70,经鉴定与预期结果一致;脾淋巴细胞杀伤活性结果显示,核酸疫苗pIRES2-MLAA34-HSP70对U937细胞的杀伤效率明显高于其他实验组及对照组(P<0.01);核酸疫苗pIRES2-MLAA34-HSP70组细胞因子IL-2、IL-4和IFN-γ水平亦明显高于其他实验组及对照组(P<0.01)。结论成功构建了pIRES2-MLAA34-HSP70核酸疫苗,该核酸疫苗能诱发强烈的体液免疫,增强机体对肿瘤细胞的免疫应答,对MLAA34阳性细胞具有特异性杀伤作用。
Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.MethodsThe acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein(HSP)70gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4and IFN-γwere also detected by using ELISA.Results The MLAA34-HSP70gene(2 956bp)and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937cells was significantly higher than that in other experimental groups and control group(P<0.01),and levels of IL-4,IL-2and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group(P<0.01).Conclusion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.
Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.MethodsThe acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein(HSP)70gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4and IFN-γwere also detected by using ELISA.Results The MLAA34-HSP70gene(2 956bp)and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937cells was significantly higher than that in other experimental groups and control group(P<0.01),and levels of IL-4,IL-2and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group(P<0.01).Conclusion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.
引文
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[10]Dahm P,Jennewein S.Introduction of the early pathway to taxol biosynthesis in yeast by means of biosynthetic gene cluster construction using SOE-PCR and homologous recombination[J].Methods Mol Biol,2010(643):145-163。
[11]张旻,姜绍通,郑娟,等.潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化[J].生物工程学报,2015,31(8):1203-1218.
[12]Yuelan Z,Yiwei L,Liyuan L,et al.Expression and identification of the ADF-linker-3-1Egene of Eimeria acervulina of chicken[J].Parasitol Res,2016,115(4):1641-1647.
[13]Li B,Ye J,Lin Y,et al.Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9[J].Vet Immunol Immunopathol,2014,161(3/4):258-264.
[2]Thornton JA.Splicing by overlap extension PCR to obtain hybrid DNA products[J].Methods Mol Biol,2016,1373:43-49.
[3]彭雷,赵艳,马银花.基于巢式PCR的重叠延伸PCR优化[J].安徽农业科学,2016,44(20):126-127.
[4]Donnelly JJ,Wahren B,Liu MA.DNA vaccines:progress and challenges[J].J Immunol,2005,175(2):633-639.
[5]Zhao J,He A,Zhang W,et al.Quantitative assessment of MLAA-34expression in diagnosis and prognosis of acute monocytic leukemia[J].Cancer Immunol Immunother,2011,60(4):587-597.
[6]Zhang WJ,Zhang WG,Zhang PY,et al.The expression and functional characterization associated with cell apoptosis and proteomic analysis of the novel gene MLAA-34in U937cells[J].Oncol Rep,2013,29(2):491-506.
[7]赵天宇,赵佳琪,范业宁,等.pIRES2-MLAA34-EGFP穿梭载体的构建及表达[J].吉林医药学院学报,2016,37(3):184-187.
[8]游朝勇.HSP70与恶性肿瘤关系的研究进展[J].实用癌症杂志,2012,27(1):89-91,94.
[9]Guzhova IV,Margulis BA.HSP70-based anti-cancer immunotherapy[J].Hum Vaccin Immunother,2016,12(10):2529-2535.
[10]Dahm P,Jennewein S.Introduction of the early pathway to taxol biosynthesis in yeast by means of biosynthetic gene cluster construction using SOE-PCR and homologous recombination[J].Methods Mol Biol,2010(643):145-163。
[11]张旻,姜绍通,郑娟,等.潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化[J].生物工程学报,2015,31(8):1203-1218.
[12]Yuelan Z,Yiwei L,Liyuan L,et al.Expression and identification of the ADF-linker-3-1Egene of Eimeria acervulina of chicken[J].Parasitol Res,2016,115(4):1641-1647.
[13]Li B,Ye J,Lin Y,et al.Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9[J].Vet Immunol Immunopathol,2014,161(3/4):258-264.