CAJ | 학술논문

目的构建表达人黑色素瘤相关抗原D4a(MAGE-D4a)基因的重组腺病毒载体并进行鉴定.方法从胶质瘤细胞系中用反转录-聚合酶链反应(RT-PCR)方法得到MAGE-D4a基因全长片段,将其导入T载体进行测序,得到准确的序列后再导入腺病毒穿梭质粒中.将穿梭质粒和病毒骨架通过电穿孔导入细菌内进行重组,挑选阳性克隆,提取重组后的质粒,用脂质体法将其在体外转染入HEK293细胞,并在细胞内部包装.扩增收集重组腺病毒Ad-MAGE-D4a,RT-PCR方法检测其体外表达,并用倍比稀释感染法测定腺病毒的滴度.结果 MAGE-D4a基因被导入带有绿色荧光的腺病毒载体中,PCR检测证实其在转染后的HEK293细胞中表达,病毒滴度为2× 108 pfu/ml.结论成功构建表达人MAGE-D4a基因的重组腺病毒载体,有效转染HEK293细胞并进行基因表达.
목적 구건표체인흑색소류상관항원D4a(MAGE-D4a)기인적중조선병독재체병진행감정.방법 종효질류세포계중용반전록-취합매련반응(RT-PCR)방법득도MAGE-D4a기인전장편단,장기도입T재체진행측서,득도준학적서렬후재도입선병독천사질립중.장천사질립화병독골가통과전천공도입세균내진행중조,도선양성극륭,제취중조후적질립,용지질체법장기재체외전염입HEK293세포,병재세포내부포장.확증수집중조선병독Ad-MAGE-D4a,RT-PCR방법검측기체외표체,병용배비희석감염법측정선병독적적도.결과 MAGE-D4a기인피도입대유록색형광적선병독재체중,PCR검측증실기재전염후적HEK293세포중표체,병독적도위2× 108 pfu/ml.결론 성공구건표체인MAGE-D4a기인적중조선병독재체,유효전염HEK293세포병진행기인표체.
Objective To construct and identify the recombinant adenovirus carrying with MAGE-D4a gene.Methods The MAGE-D4a sequencing was performed by reverse transcription RNA (RT-PCR) from Glioma cell line U251,and inserted the adeuoviral shuttle vector-pTracer-GFP.Then it was linearized with PmeI and cotransformed with E1-deleted adenoviral backbone AdEasy-1 into the competent bacterial strain BJ5183,which allows efficient recombination to occur.After screening,recombinants for adenoviruses Ad/MAGE-D4a and Ad-GFP,which contains no insert sequence as a control,were generated.The recombinant DNA was identified by restriction endonuclease PacⅠ and transfected into HEK 293 cells after linearization.The cells were harvested when a cytopathic effect appeared,and the generated recombinant adenoviruses were isolated.Results According to the result of RT-PCR,the MAGE-D4a gene was expressed by the recombinant adenovirus and the titers were 2×108 pfu/ml.Conclusions The recombinant adenovirus Acl/MAGE-D4a was successfully constructed,and the establishment of stalbly transfected HEK293 cell lines provided an original method for oncotherapy with MAGE-D4a gene.