核酸疫苗p EGFPN1-MLAA34-B7H4IgV的构建及其免疫活性观察

Construction of DNA vaccine pEGFPN1-MLAA34-B7H4IgV and its immune activity

目的构建p EGFPN1-MLAA34-B7H4IgV核酸疫苗,并检测其免疫活性,探讨核酸疫苗抗白血病效应。方法采用重叠延伸PCR技术扩增急性白血病相关抗原基因MLAA34和负性共刺激分子B7H4的融合基因(MLAA34-B7H4IgV),并构建核酸疫苗p EGFPN1-MLAA34-B7H4IgV。将28只BALB/c小鼠随机分为P、M、B、MB组各7只,分别在小鼠两侧股四头肌内侧肌内注射磷酸盐缓冲液p EGFPN1、p EGFPN1-MLAA34、p EGFPN1-B7H4IgV、p EGFPN1-MLAA34-B7H4IgV质粒100μg,在0、2、4周各行1次同剂量加强免疫;分别于0、4、8周眼球取血,8周取血后处死小鼠并摘取脾脏。ELISA法检测免疫8周血清中抗体Ig G1、Ig G2a以及免疫0、4、8周血清细胞因子IL-2、IL-4、IFN-γ,MMT法检测小鼠脾淋巴细胞对单核巨噬细胞THP-1的增殖抑制作用。结果 MLAA34-B7H4IgV融合基因PCR产物电泳检测约为2 000 bp,与理论值(1 922 bp)相符; p EGFPN1-MLAA34-B7H4IgV酶切后电泳检测为5 000~7 500 bp,与理论值(6 655 bp)相符。与同组免疫0周时、同时点P组比较,免疫4、8周时M、B、MB组血清细胞因子IL-2、IL-4水平升高而IFN-γ水平降低(P均<0. 05或<0. 01)。与同时点M、B组比较,MB组血清细胞因子IL-2、IL-4水平升高而IFN-γ水平降低(P均<0. 05); M、B组同时点比较,差异无统计学意义。免疫8周,MB组血清Ig G1水平高于其他各组(P均<0. 05),其他各组间差异均无统计学意义(P均> 0. 05);各组血清Ig G2a水平差异均无统计学意义(P均> 0. 05)。MB组THP-1细胞增殖抑制率高于同效靶比其他各组(P均<0. 05或<0. 01);效靶比为50∶1和25∶1时,M组和B组THP-1细胞增殖抑制率高于高于P组(P均<0. 05),两组之间无差异;效靶比为12. 5∶1时,B组THP-1细胞增殖抑制率高于M组、P组(P均<0. 05)。结论成功构建了核酸疫苗p EGFPN1-MLAA34-B7H4IgV,该疫苗激活了小鼠体内的细胞免疫和体液免疫,具有明显的抗白血病效应。

Objective To construct the DNA vaccine pEGFPN1-MLAA34-B7H4IgV,and detect its immune activity and anti-leukaemia effect.Methods MLAA34-B7H4IgV fusion gene was amplified by SOE-PCR and the DNA vaccine pEGFPN1-MLAA34-B7H4IgV was constructed.Then 28 BALB/c mice were randomly divided into P,M,B and MB groups,with 7 mice in each group.The mice were immunized with 100μg plasmid pEGFPN1,pEGFPN1-MLAA34,pEGFPN1-B7H4IgV,and pEGFPN1-MLAA34-B7H4IgV at 0,2nd and 4th weeks.Blood samples were collected at 0,2nd,and 4th weeks,spleen samples were taken after collecting blood samples at 8th weeks.The cytokine levels of IL-2,IL-4,and IFN-γwere detected at 0,2nd and 4th weeks,and the antibody levels of IgG1,IgG2a were detected after blood samples collection at 8th weeks by ELISA.The inhibitory effect of spleen lymphocytes on proliferation of mononuclear macrophage THP-1 was detected by using MTT.Results The electrophoresis of MLAA34-B7H4IgV fusion gene PCR product was about 2 000 bp,which was consistent with the theoretical value(1 922 bp).After enzyme digestion,the electrophoresis of pEGFPN1-MLAA34-B7H4IgV plasmid was between 5 000 bp and 7 500 bp,which was consistent with the theoretical value(6 655 bp).Compared with the same group at immunization of 0 weeks or compared with group P at the same time point,the levels of serum cytokines IL-2 and IL-4 increased while the levels of IFN-γdecreased in the M,B and MB groups at 4 and 8 weeks after immunization(P<0.05 or P<0.01).Compared with the groups M and B at the same time point,the levels of cytokines IL-2 and IL-4 in the MB group increased,while the level of IFN-γdecreased(P<0.05),and there was no significant difference between group M and group B at the same time point(P>0.05).After 8 weeks of immunization,serum IgG1 level in the group MB was higher than that in the other groups(P<0.05)and there was no significant difference between the other groups(P>0.05).There was no significant difference in the level of serum IgG2a between every two groups(P>0.05).The inhibition rate of THP-1 cells in the group MB was higher than that at the same target ratio of the other groups(P<0.05 or P<0.01).When the target ratio was 50∶1 and 25:1,the inhibition rate of THP-1 cells in the groups M and B was higher than that in the group P(P<0.05),and there was no difference between the two groups.When the target ratio was 12.5∶1,the inhibition rate of THP-1 cells in the group B was higher than that in the groups M and P(P<0.05).Conclusion DNA vaccine pEGFPN1-MLAA34-B7H4IgV is constructed successfully.The DNA vaccine activates the humoral immunity and cellular immunity and has an significant anti-leukemic effect.