含不同启动子的人癌胚抗原重组质粒的构建与表达

Construction and Expression of Carcinoembryonic Antigen Recombinant Plasmids with Two Different Promoters

构建含不同启动子的人癌胚抗原重组表达质粒,观察肌肉注射该重组质粒后人癌胚抗原基因在小鼠舌肌中的表达情况。方法:将7天前肌肉注射0.75%布比卡因的小鼠分成4组,每组4只,分别将含不同启动子的重组质粒pCEA、pLCEASN及阴性对照质粒pcDNA3、pLXSN注射于小鼠的舌肌内,每周1次,每次100μg,共3次;注射前及每次注射后2天,取尾静脉血用放免法测其癌胚抗原的含量,最后1次注射2天后,处死小鼠并取其舌、脑、肝、脾、肾、心脏及股四头肌用免疫组化法(ABC法)检测癌胚抗原的表达。结果:构建并鉴定了两种新的癌胚抗原重组表达质粒;免疫组织化学法检测显示:癌胚抗原ABC染色仅在用人癌胚抗原重组表达质粒注射过的舌肌切片中阳性,而在脑、肝、脾、肾、心脏及股四头肌的切片中均阴性;放免法检测显示:所有血液样本中癌胚抗原的浓度均未达检测值(<5ng/ml)。结论:含不同启动子的癌胚抗原重组表达质粒可在注射部位有效地表达人癌胚抗原。

Objective:1)To construct two carcinoembryonic antigen(CEA)recombinant ex-pression plasmids,in which the full length complementary DNA(cDNA)for human CEA is under the con trol of different promoters;2)To detect the carcinoembryonic antigen expressed in mice tongues by direct intramuscular injec tion of these CEA recombinant expression plasmids.Methods:At first,molecular cloning techniques were applied to construct two recombinant expression plas-mids,in which the human CEA cDNA was driven by CMV promoter /enhancer(pCEA)or LTR(pLCEASN),respectively.Groups of mice(n=4),which were in tra muscular injected with0.75%bupivacaine7days ago,were in tra muscular injected with pCEA?pLCEASN?pcDNA3or pLXSN,100μg does week ly for three injections,respectively.The sera from tailer vein were harvested at proinjection and each2days postin jection for radio-immunology assay.All mice were sacrificed2days after the last injection,and the tongue?brain?liver?kidney?spleen?heart and quadricep muscle were re moved for CEA im munohisto chemical staining(ABC method).Results:1)Two car-cinoem bry on ic anti gen re combinant expression plasmids were con structed and identified;2)ABC staining for CEA were positive in sections of the tongue injected with plasmid DNA of pCEA?pLCEASN except pcDNA3and pLXSN,but negative in the brain?liver?kidney?spleen?heart and quadricep muscle;3)The CEA con centrations were under the detectable CEA level(<5ng /ml)by ra dio-im munolo gy assay in all sera samples.Con clusion:Human CEA cDNA can be ex-pressed effectively in situ after injection of these CEA re combi nant expression plasmids,in which the human CEA cD NA is driv en by different promoters.