摘要
PCR扩增嗜肺军团菌mip基因和霍乱弧菌ctxB基因,克隆入载体pcDNA3 1( +),重组子经限制性酶切分析、PCR、序列分析鉴定正确后,命名为pcDNA3 1 mip/ctxB.脂质体法将重组质粒pcDNA3 1 mip、pcDNA3 1 mip/ctxB转染NIH3T3细胞,用免疫荧光法和蛋白质印迹鉴定瞬时表达和稳定表达产物,结果发现:重组质粒成功转入细胞并获得短暂表达,稳定转染细胞分别在 2 4ku和 35ku处检测到阳性杂交信号.将pcDNA3 1 mip、pcDNA3 1 mip/ctxB作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFN γ产生水平、细胞毒性T淋巴细胞(CTL)杀伤活性等体液免疫和细胞免疫反应的指标,评价疫苗的免疫原性.结果发现:各实验组均检测到免疫原性,pcDNA3 1 mip/ctxB免疫组的免疫原性高于pcDNA3 1 mip免疫组,有显著性差异(P <0 0 1).
Mip gene of Legionella pneumophila and ctxB gene of Vibrio cholerae were PCR amplified respectively. The amplified fusion DNA was ligated to pcDNA3. 1 (+) vector. The recombinant plasmid, which was named pcDNA3. 1-mip/ctxB, was identified by restriction analysis, PCR and further confirmed by sequence analysis. NIH3T3 cell was transfected by recombinant plasmid pcDNA3. 1-mip or pcDNA3. 1-mip/ctxB with Lipofection strategy. Transient and stable products of mip/ctxB fusion gene was detected by immunofluorescence and Western blot. The results showed that NIH3T3 cell was transfected successfully and stable products can be detected in the transfected cell. To evaluate immunonicity of pcDNA3. 1-mip and pcDNA3. 1-mip/ctxB, BALB/c female mice were immunized intramuscularly with them and antigen specific antibodies, lymphocyte proliferative response, IFN-gamma production and cytotoxic T-lymphocyte response of immunized mice were detected. The results showed that immunogenicity of pcDNA3. 1-mip or pcDNA3. 1-mip/ctxB immunized mice were higher than control and immunogenicity of peDNA3. 1-mip/ctxB immunized mice were higher than pcDNA3. 1-mip immuneized mice. Statistic analysis by one way ANOVA showed that there was significantly difference between groups (P < 0.01). The results provide experimental proof to study of mip/ctxB fusion gene DNA vaccine.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2004年第9期818-823,共6页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(3 0 3 0 0 3 0 2)~~
