Co-expression and Immunity of Legionella pneumophila mip Gene and Immunoadjuvant ctxB Gene  

作　　者：TaoWANG Jian-PingCHEN HongLI Ke-QianZHI LeiZHANG Chun-LeiYANG Da-ChangTAO 

机构地区：[1]SchoolofPreclinicalandForensicMedicineofWestChina,SichuanUniversity,Chengdu610041,China [2]SchoolofStomatologvXi＇anJiaotongUniversity,Xi＇an710004,China [3]SchoolofLifeScience,SichuanUniversity,Chengdu610041,China

出　　处：《Acta Biochimica et Biophysica Sinica》2005年第3期199-204,共6页生物化学与生物物理学报（英文版）

基　　金：This work was supported by a grant from the National Natural Science Foundation of China(No.30300302)

摘　　要：The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively.The amplified cDNA was ligated to the pcDNA3.1(+)vector.The recombinant plasmids pcDNA3,1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR,and further confirmed by sequencing analysis.NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method.Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting.The results showed that NIH3T3 cells were successfully transfected,and that the transiently and stably co-expressed products can be detected in the transfected cells.To detect the humoral and cellular immune response in immunized mice induced by the co- immunization of the mip and ctxB genes,female BALB/c mice were immunized intramuscularly with pcDNA3.1- mip and pcDNA3.1-ctxB.The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3,1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization.Furthermore,the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization.Statistical analysis using one-way analysis of variance(ANOVA)showed that there was a significant difference between the groups(P<0.01).The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization.These findings provide experimental evidence to support the development of the L.pneumophila DNA vaccine.The mip gene of Legionella pneumophila and the ctxB gene of Vibrio cholerae were amplified by PCR respectively.The amplified cDNA was ligated to the pcDNA3.1(+)vector.The recombinant plasmids pcDNA3,1-mip and pcDNA3.1-ctxB were identified by restriction analysis and PCR,and further confirmed by sequencing analysis.NIH3T3 cells were transfected with pcDNA3.1-mip and pcDNA3.1-ctxB according to the Lipofection method.Transient and stable products of the co-expression of the mip gene and ctxB gene were detected by immunofluorescence and Western blotting.The results showed that NIH3T3 cells were successfully transfected,and that the transiently and stably co-expressed products can be detected in the transfected cells.To detect the humoral and cellular immune response in immunized mice induced by the co- immunization of the mip and ctxB genes,female BALB/c mice were immunized intramuscularly with pcDNA3.1- mip and pcDNA3.1-ctxB.The results showed that the specific antibody titer and the cytotoxic T-lymphocyte response for pcDNA3,1-mip immunization and co-immunization were increased compared with that of pcDNA3.1(+) immunization.Furthermore,the specific antibody titer and cytotoxic T-lymphocyte response for co-immunization were increased compared with that of pcDNA3.1-mip immunization.Statistical analysis using one-way analysis of variance(ANOVA)showed that there was a significant difference between the groups(P<0.01).The results indicated that the ctxB gene enhanced the humoral and cellular immune response to the mip gene immunization.These findings provide experimental evidence to support the development of the L.pneumophila DNA vaccine.

关 键 词：Legionella pneumophila CO-EXPRESSION humoral immunity cellular immunity 

分 类 号：R392.2[医药卫生—免疫学]