CAJ | 학술논문

为构建单纯疱疹病毒I型胸苷激酶(HSV1TK)的真核表达载体pcDNA3.1-EGFP/HSV1TK,鉴定其在真核细胞中的表达和功能.以pORF-HSV1TK为模板,PCR扩增的目的基因HSV1TK片段与pMD18-T载体相连接构建重组克隆pMD18-T/HSV1TK.再双酶切出HSV1TK片段,插入pcDNA3.1-EGFP多克隆位点,构建pcDNA3.1-EGFP/ HSV1TK真核表达载体并进行酶切、测序鉴定[1].分别用荧光显微镜观察和RT-PCR方法检测脂质体介导pcDNA3.1-EGFP/ HSV1TK在卵巢癌细胞SKOV3的表达;分别用MTT法和光镜检测胸苷激酶/丙氧鸟苷(HSV1TK/GCV)系统对SKOV3体外杀伤作用及旁观者效应.结果表明,重组载体酶切鉴定结果与预期结果一致,基因序列与GenBank上报道的HSV1TK基因序列完全一致.荧光显微镜观察转染后的细胞发出绿色荧光;RT-PCR结果表明HSV1TK基因能在SKOV3内有效表达.MTT和光镜结果显示转染HSV1TK基因的SKOV3细胞,加入前体药物丙氧鸟苷(GCV)处理后对其有明显的杀伤作用和旁观者效应.成功构建的真核表达载体pcDNA3.1-EGFP/ HSV1TK能在SKOV3细胞中稳定表达,且HSV1TK对卵巢癌细胞株SKOV3体外有强大的杀伤作用和旁观者效应.
위구건단순포진병독I형흉감격매(HSV1TK)적진핵표체재체pcDNA3.1-EGFP/HSV1TK,감정기재진핵세포중적표체화공능.이pORF-HSV1TK위모판,PCR확증적목적기인HSV1TK편단여pMD18-T재체상련접구건중조극륭pMD18-T/HSV1TK.재쌍매절출HSV1TK편단,삽입pcDNA3.1-EGFP다극륭위점,구건pcDNA3.1-EGFP/ HSV1TK진핵표체재체병진행매절、측서감정[1].분별용형광현미경관찰화RT-PCR방법검측지질체개도pcDNA3.1-EGFP/ HSV1TK재란소암세포SKOV3적표체;분별용MTT법화광경검측흉감격매/병양조감(HSV1TK/GCV)계통대SKOV3체외살상작용급방관자효응.결과표명,중조재체매절감정결과여예기결과일치,기인서렬여GenBank상보도적HSV1TK기인서렬완전일치.형광현미경관찰전염후적세포발출록색형광;RT-PCR결과표명HSV1TK기인능재SKOV3내유효표체.MTT화광경결과현시전염HSV1TK기인적SKOV3세포,가입전체약물병양조감(GCV)처리후대기유명현적살상작용화방관자효응.성공구건적진핵표체재체pcDNA3.1-EGFP/ HSV1TK능재SKOV3세포중은정표체,차HSV1TK대란소암세포주SKOV3체외유강대적살상작용화방관자효응.
It was construct EGFP and HSV1TK co-expression vector and detect its expression and function in eukaryocyte ovarian cancer cells SKOV3. The HSV1TK gene were inserted into cloning vector pMD18-T to construct the plasmid of pMD18/ HSV1TK.The HSV1TK gene fragment obtained from pMD18T/HSV1TK that was digested,and then inserted into pcDNA3.1-EGFP. The recombinant pcDNA3.1-EGFP/ HSV1TK was identified with restriction analysis and DNA sequence. The expression plasmid pcDNA3.1-EGFP/ HSV1TK was transfected into ovarian cancer cells SKOV3 mediated by liposome reagent, then the expressions of EGFP in cells were observed by fluorescence microscopy and HSV1TK was detected with RT-PCR. The killing SKOV3 effect and bystander effect of HSV1TK/GCV was observed by MTT and light microscope. Results showed that the sequence of the cloned DNA fragment was identical to HSV1TK that was reported on GenBank, and the expression of vector pcDNA3.1-EGFP was correct. The recombinant expression plasmid was successfully transferred into ovarian cancer cells SKOV3 , and effective expression of HSV1TK was also testified by RT-PCR. Thus,the recombinant eukaryotic co-expression vector of EGFP and HSV1TK was successfully constructed and effectively expressed in ovarian cancer cells SKOV3. HSV1TK/GCV has better killing effects and bystander effect in SKOV3 cells .