中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
3期
330-332
,共3页
韩从辉%郝林%胡建鹏%王涛%贡震%贺厚光%董秉政%张培影
韓從輝%郝林%鬍建鵬%王濤%貢震%賀厚光%董秉政%張培影
한종휘%학림%호건붕%왕도%공진%하후광%동병정%장배영
前列腺癌%前列腺特异性抗原%端粒酶逆转录酶%超抗原SEA%溶瘤腺病毒
前列腺癌%前列腺特異性抗原%耑粒酶逆轉錄酶%超抗原SEA%溶瘤腺病毒
전렬선암%전렬선특이성항원%단립매역전록매%초항원SEA%용류선병독
Prostatic carcinoma%Prostate specific antigen%Telomerase reverse transeriptase%Superanfigen SEA%Oncolytic adenovirub
目的 构建表达超抗原金黄色葡萄球菌肠毒素(SEA)基因的由前列腺特异性抗原(PSA)启动子及端粒酶逆转录酶(hTERT)启动子双调控的特异性增殖溶瘤腺病毒SG504-SEA.方法 从前列腺癌组织基因组DNA中获得522 bp大小的PSA启动子序列,将PSA启动子克隆到由hTERT启动子调控的特异性增殖溶瘤腺病毒载体SG502中.得到靶向前列腺癌细胞的双调控溶瘤腺病毒载体SG504.将已构建好的含有771 bp大小SEA基因片段的病毒骨架质粒PPE3-cdb-SEA与SG504用Lipofectamine 2000共转染至293细胞.共转染后9～14 d出现病毒空斑,经过3次病毒空斑纯化,经鉴定正确的腺病毒命名为SG504.SEA,即携带SEA基因的双调控靶向前列腺癌的特异性溶瘤腺病毒.结果 经聚合酶链反应(PCR)及酶切鉴定,SEA基因成功克隆到病毒载体中,可以表达SEA基因,且病毒滴度为2.0×10~(10)pfu/ml.结论 成功构建表达超抗原SEA基因的双调控选择增殖型溶瘤腺病毒SG504-SEA.
目的 構建錶達超抗原金黃色葡萄毬菌腸毒素(SEA)基因的由前列腺特異性抗原(PSA)啟動子及耑粒酶逆轉錄酶(hTERT)啟動子雙調控的特異性增殖溶瘤腺病毒SG504-SEA.方法 從前列腺癌組織基因組DNA中穫得522 bp大小的PSA啟動子序列,將PSA啟動子剋隆到由hTERT啟動子調控的特異性增殖溶瘤腺病毒載體SG502中.得到靶嚮前列腺癌細胞的雙調控溶瘤腺病毒載體SG504.將已構建好的含有771 bp大小SEA基因片段的病毒骨架質粒PPE3-cdb-SEA與SG504用Lipofectamine 2000共轉染至293細胞.共轉染後9～14 d齣現病毒空斑,經過3次病毒空斑純化,經鑒定正確的腺病毒命名為SG504.SEA,即攜帶SEA基因的雙調控靶嚮前列腺癌的特異性溶瘤腺病毒.結果 經聚閤酶鏈反應(PCR)及酶切鑒定,SEA基因成功剋隆到病毒載體中,可以錶達SEA基因,且病毒滴度為2.0×10~(10)pfu/ml.結論 成功構建錶達超抗原SEA基因的雙調控選擇增殖型溶瘤腺病毒SG504-SEA.
목적 구건표체초항원금황색포도구균장독소(SEA)기인적유전렬선특이성항원(PSA)계동자급단립매역전록매(hTERT)계동자쌍조공적특이성증식용류선병독SG504-SEA.방법 종전렬선암조직기인조DNA중획득522 bp대소적PSA계동자서렬,장PSA계동자극륭도유hTERT계동자조공적특이성증식용류선병독재체SG502중.득도파향전렬선암세포적쌍조공용류선병독재체SG504.장이구건호적함유771 bp대소SEA기인편단적병독골가질립PPE3-cdb-SEA여SG504용Lipofectamine 2000공전염지293세포.공전염후9～14 d출현병독공반,경과3차병독공반순화,경감정정학적선병독명명위SG504.SEA,즉휴대SEA기인적쌍조공파향전렬선암적특이성용류선병독.결과 경취합매련반응(PCR)급매절감정,SEA기인성공극륭도병독재체중,가이표체SEA기인,차병독적도위2.0×10~(10)pfu/ml.결론 성공구건표체초항원SEA기인적쌍조공선택증식형용류선병독SG504-SEA.
Objective To construct double-regulated conditionally replicating adenovirus SG504SEA.Methods The prostate-specific antigen(PSA)promoter was amplified from prostate cancer tissues by polymerase chain reaction(PCR)method and the 522 bp PSA promoter gene sequence was cloned into pSC504 plasmids after restriction enzyme cutting.The plasmid pSG504 was co-transfected with PPE3-ccdbSEA in 293 cells to generate recombinant adenoviruses SG504-SEA.The recombinant adenoviruses wereverified by PCR.purified by cesium chlofide density purification and propagated in 293 cells.Vires titer was measured by TCID50 methed and its titre was 2.0×10~(10) pfu/m1.Results It was proven that the successful cloning of PSA promoter and SEA gene into the oncolytic adenovirus vector could realize the expres-sion of the SEA gene.And the virus titer was 2.0×10~(10)pfu/ml.Conclusion We have successfully constmeted double-regulated conditionally replicating adenovirus SG504-SEA.which lavs the foundation for further research on application of SEA in targeted gene therapy for prostate cancer and the underlying immunological mechanisms.
