SEA和喉癌MAGE-A3构建的真核质粒pMAGEA3-IRES-SEA经过电穿孔转染在小鼠体内的转录

The transcription of eukaryotic coexpression vector pMAGEA3-IRES-SEA of recombinant MAGE-A3 originating from laryngocarcinoma and staphylococcal endotoxin A after electroporation in mice in vivo

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.