摘要
目的检测真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠中的转录。方法将葡萄球菌肠毒素A(staphylococcal endotoxin A,SEA)和喉癌来源的黑色素瘤抗原A3(melanomaassociated antigen A3,MAGE-A3),构建成基因共表达的真核质粒pMAGEA3-IRES-SEA。将pMAGEA3-IRES-SEA用电转染的方法免疫BALB/C小鼠单侧股四头肌,每2周1次,每次注射50μg,共免疫3次。末次免疫2周后,取注射部位组织,用荧光定量PCR(qRT-PCR)法检测目的基因在注射部位肌肉中的转录情况。结果在实验小鼠注射部位的骨骼肌内检测到SEA、MAGE-A3基因转录mRNA。结论所构建的pMAGEA3-IRES-SEA真核表达质粒,可通过电转染的方式,在小鼠体内能有效地转录。这为研究该质粒通过小鼠的体内转录蛋白的表达,诱导机体细胞免疫、体液免疫来清除喉癌,奠定了理论基础。
Objective To identify the transcription of eukaryotic coexpression vector pMAGEA3-IRES-SEA by electroporation method in muscle of mice. Methods The eukaryotic coexpression vector was constructed with staphylococcal endotoxin A（SEA） and human melanoma-associated antigen gene A3 （MAGE-A3） originating from laryngocarcinoma by genetic recombinant method in advance. Unilateral hindlimb skeletal muscles of BALB/C mice were immuned with eukaryotic expression plasmid pMAGEA3-IRES-SEA by electrotransfection method,one time every two week, each immuned of 50μg, a total of three times. Two weeks after the final immunization, their gene transcriptions were identified at the muscle organization of the injection site by fluorescence quantitative PCR （qRT-PCR） assay. Results The SEA and MAGE-A3 genes had been transcripted in experimental mice skeletal muscle. Conclusions The constructed pMAGEA3-IRES-SEA eukaryotic expression plasmid can be effectively transcripted in mice by electroporation method. This work provided a theoretical basis for the expression of mouse in vivo transcription protein and the application of the plasmid to clear laryngocarcinoma by inducing the cellular immunity and humoral immunity in organism.
出处
《中华临床医师杂志（电子版）》
CAS
2014年第1期86-90,共5页
Chinese Journal of Clinicians(Electronic Edition)
基金
广东省科技厅产业技术研究与开发资金计划项目(2012B031800340)
广州市科技和信息化局社会发展应用基础研究专项(2013J4100024)
关键词
葡萄球菌属
超抗原
黑色素瘤
电穿孔
喉肿瘤
电转染
疫苗,DNA
Staphylococcus
Superantigen
Melanoma
Vaccine,DNA
Electroporation
Laryngeal carcinoma
Electrotransfection
