嗜肺军团菌momp基因疫苗的制备及其免疫原性研究  

Preparation and immunogenicity of mompgene vaccine of Legionella Pneumophilia

作　　者：窦娇莹[1,2] 杨志伟[2] 

机构地区：[1]甘肃省中医院检验科,兰州730050 [2]宁夏医科大学基础医学院病原生物学与免疫学系,银川750004

出　　处：《国际检验医学杂志》2016年第11期1449-1452,共4页International Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目(30860264)

摘　　要：目的构建真核重组质粒GFP-momp作为核酸疫苗,已构建的原核重组质粒pET-momp表达的蛋白作为蛋白疫苗,二者联合免疫BALB/c雌性小鼠,观察其免疫原性。方法 (1)以嗜肺军团菌DNA作为模板,扩增得到momp基因,并克隆至pEGFP-C1载体获得重组质粒GFP-momp。鉴定正确后,将其转染到NIH3T3细胞,利用荧光显微镜观察GFP-momp的表达。(2)选取BALB/c雌性小鼠60只,平均分为4组,即磷酸盐缓冲液(PBS)组(A组)、空质粒pEGFP-C1组(B组)、DNA疫苗组(C组)、联合疫苗组(D组)。以重组质粒GFP-momp作为DNA疫苗,重组质粒GFP-momp和MOMP蛋白作为联合疫苗,第1天A组肌肉注射PBS 50μL,B组注射pEGFP-C1 50μg,C、D组各注射GFP-momp50μg;第14天各组以相同剂量追加免疫1次;第21天A、B、C组用相同剂量再追加免疫1次,D组注射10μg纯化的MOMP蛋白。借助对各试验组小鼠的抗体水平、脾淋巴细胞增殖活性、细胞毒性T淋巴细胞(CTL)杀伤活性等指标的动态检测,来评价DNA疫苗与蛋白疫苗的免疫原性。结果扩增出831bp的momp基因,构建重组质粒GFP-momp,转染入NIH3T3细胞并在细胞内表达出绿色荧光蛋白。在淋巴细胞增殖试验中:A组和B组比较差异无统计学意义(P>0.05);C组和D组淋巴细胞增殖活性均高于B组,差异有统计学意义(P<0.05);C组高于D组,差异有统计学意义(P<0.05)。间接ELISA测定血清中抗原特异性抗体水平结果显示,A、B两组比较差异无统计学意义(P>0.05);C、D两组均高于B组,差异有统计学意义(P<0.05);D组抗体滴度高于C组,差异有统计学意义(P<0.05)。CTL杀伤活性试验结果显示,A、B两组比较差异无统计学意义(P>0.05);C、D两组均高于B组,差异有统计学意义(P<0.05);D组杀伤活性高于C组,差异有统计学意义(P<0.05)。结论成功构建momp基因的真核表达载体并在NIH3T3细胞中得到表达,并进一步得出嗜肺军团菌momp基因诱导产生的DNA疫苗和DNA-蛋白疫苗均能产生细胞免�Objective To observe the immunogenicity of the DNA vaccine induced by eukaryotic recombinant plasmid GFPmompcombined with protein vaccine induced by prokaryotic recombinant plasmid pET-mompin BALB/c female mice immunized intramuscularly.Methods The MOMP gene amplified from DNA of Legionella pneumophila by PCR were cloned into pEGFP-C1 vector,which produced the recombinant plasmid named as GFP-momp.GFP-momp was analyzed with restriction endonuclease digestion,PCR and DNA sequencing techniques.The NIH3T3 cell was transfected by recombinant plasmid GFP-mompthrough lipofection.The stable expression products of MOMP were observed by the fluorescent microscope.Sixty BALB/c female mice were divided into 4groups averagely,the PBS control group（A group）,the plasmid pEGFP-C1 control group（B group）,DNA vaccine experimental group（C group）and the combination vaccine experimental group（D group）.By recombinant plasmid the GFP-momp combined with MOMP protein,mice of group A were given intramuscular injection PBS 50μL and mice of group B were injected with pEGFP-C1 50μg.In addition,mice of group C and D were injected GFP-momp50μg in the first day.Each group injected the same dose immunization once in the fourteenth day.Then mice of group A,B and C were injected with the same dose of an additional immunization and in the twenty-first day and that of the group D were injected with 10μg purified MOMP protein.Then antibody levels,lymphocyte proliferation activity and CTL killing activity were tested to evaluate the immunogenicity of DNA vaccines and combined vaccine.Results 831 bp mompgene was amplified.Under the fluorescent microscope,green fluorescent was observed in the cell cytoplasm.In the lymphocyte proliferation test,there was no significant difference between group A and B（P〉0.05）.Proliferative activity of group C and D were significantly higher than that of group B（P〈0.05）.And proliferative activity of group C was significantly higher than that of group D（P〈0.05）.The indirect ELISA resul

关 键 词：嗜肺军团菌 momp基因 疫苗 

分 类 号：R392-33[医药卫生—免疫学]