基于SOE-PCR的核酸疫苗pIRES2-MLAA34-HSP70的构建及免疫效果研究

Study on immune effect of DNA vaccine pIRES2-MLAA34-HSP70 constructed by SOE-PCR

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 plRES2-MLAA34-HSP70,and to detect its immune effect. Methods The acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein （HSP）70 gene 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 plRES2-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-4 and IFN-], were also detected by u- sing ELISA. Results The MLAA34-HSP70 gene （2 956 bp） and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully. The killing efficiency of DNA vaccine pIRES2-MLAA34-HSPT0 in U937 cells was significantly higher than that in other experimental groups and control group （P〈0. 01） ,and levels of IL-4, IL-2 and IFN-7 in DNA vaccine pIRES2- MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group （P〈0.01）. Con- clusion 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.