摘要
目的构建含日本血吸虫相对分子质量为26 000的抗原(Sj26GST)基因的膜锚定表达DNA疫苗,观察其免疫BALB/c小鼠后的免疫应答效应。方法用RT-PCR法,以血吸虫成虫RNA为模板,扩增获得Sj26的全长基因。利用重组PCR技术,在Sj26基因的5′端加上编码IL-2的信号肽核苷酸序列,3′端加上编码胎盘碱性磷酸酶的膜锚定序列,然后将其克隆入pIRES载体中,构建一个膜锚定型真核表达质粒pIRES-Sj26。将重组质粒转染HeLa细胞,通过RT-PCR及间接免疫荧光技术检测目的基因的表达。用构建的pIRES-Sj26疫苗肌肉注射免疫BALB/c小鼠后,以ELISA试剂盒检测小鼠血清中的总IgG抗体浓度,脾细胞培养法检测脾淋巴细胞培养上清的干扰素γ(INF-γ)含量,淋巴细胞刺激指数(SI)反映淋巴细胞增殖能力,流式细胞术检测脾细胞CD4/CD8亚群。结果经过酶切鉴定、PCR及测序证实重组质粒pIRES-Sj26构建成功,经转染HeLa细胞及免疫荧光检测证明质粒pIRES-Sj26能在体外进行表达。免疫小鼠后检测结果表明pIRES-Sj26组的血清总IgG抗体浓度、INF-γ的含量明显高于空白对照组和空载体组(均P<0.01);其脾SI高于空白对照组和空载体组(P<0.05);CD8+细胞百分比高于空白对照组和空载体组(均P<0.05),CD4+细胞百分比没有显著变化(P>0.05)。结论成功构建日本血吸虫膜锚定表达DNA疫苗pIRES-Sj26,表达的Sj26蛋白大部分锚定在细胞膜上。pIRES-Sj26疫苗能增强BALB/c小鼠的免疫应答反应。
Objective To construct a eukaryotic membrane-anchored expression plasmid containing Sj26GST genes and determine its immunogenicity in BALB/c mice. Methods Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worm as the template and modified with a nucleotide sequence of human interleukin-2 （IL-2） signal peptides in their 5＇ ends and that of membrane-anchored peptides of carboxyl-terminal of placental alkaline phosphatase （PLAP） in 3＇ ends. Then they were cloned into eukaryotic expression plasmid pIRES to construct a recombinant plasmid pIRES-Sj26. The expression of modified gene was detected by using RT-PCR and IFA when the recombinant plasmid pIRES-Sj26 was transfected into eukaryotic HeLa cells. After immunization of BALB/c mice with pIRES-Sj26 vaccine, total IgG in serum and the level of IFN-γ were measured by ELISA. The lymphocyte stimulating index （SI） and the subgroups of splenocytes by FCM were tested also. Results Restriction enzyme analysis, PCR and sequencing results showed that the recombinant plasmid pIRES-Sj26 was constructed successfully. After the transfection, the expression of modified Sj26 genes could be detected by RT-PCR and IFA. The IgG level in the pIRES-Sj26 group was significantly higher than that in the control group and the vector group （P〈0.01）. The INF-γ level in the experimental group was also significantly higher than other groups （P〈0.01）, while the SI level was higher too （P%0.05）. Compared with the control group and the vector group, the percentages of CD8^＋ was significantly elevated in the plRES-Sj26 group （P〈0.05）, but no significant difference in the percentages of CD4^＋ was found （P〉0.05）. Conclusion A membrane-anchored DNA vaccine encoding pIRES-Sj26 was successfully constructed and Sj26 membrane-anchored proteins were proved to be expressed in vitro. The pIRES-Sj26 vaccine could induce stronger immune response in BALB/c mice.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2007年第2期141-144,152,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30471603)
