CAJ | 학술논문

目的构建大鼠胸腺素β4 (Tβ4)基因的重组慢病毒载体,转染293T细胞并观察Tβ4的表达.方法从Genbank获取大鼠Tβ4基因序列,化学合成后连接入线性化慢病毒载体pGC-FU,得到重组慢病毒载体pGC-FU-Tβ4,将其转化细菌感受态细胞后行菌落聚合酶链反应(PCR)鉴定,对PCR鉴定阳性的克隆进行测序鉴定. pGC-FU- Tβ4、pHeIper 1.0、pHelper 2.0共转293T细胞,收集上清液浓缩后使用实时定量PCR(Real-time PCR)进行滴度检测.用所得病毒感染293T细胞后,Western blot检测Tβ4表达.结果目的基因质粒酶切后琼脂糖凝胶电泳图谱示酶切片段大小为143 bp；菌落PCR结果示阴性转化子得到198 bp片段,阳性转化子得到333 bp片段；测序结果与预期相符合；Real-time PCR示所得病毒滴度约为2×109TU:Western blot示Tβ4转染组在33 KD处有特征条带.结论成功构建大鼠Tβ4基因慢病毒载体pGC-FU- Tβ4,并建立慢病毒过表达系统.
목적 구건대서흉선소β4 (Tβ4)기인적중조만병독재체,전염293T세포병관찰Tβ4적표체.방법 종Genbank획취대서Tβ4기인서렬,화학합성후련접입선성화만병독재체pGC-FU,득도중조만병독재체pGC-FU-Tβ4,장기전화세균감수태세포후행균락취합매련반응(PCR)감정,대PCR감정양성적극륭진행측서감정. pGC-FU- Tβ4、pHeIper 1.0、pHelper 2.0공전293T세포,수집상청액농축후사용실시정량PCR(Real-time PCR)진행적도검측.용소득병독감염293T세포후,Western blot검측Tβ4표체.결과 목적기인질립매절후경지당응효전영도보시매절편단대소위143 bp；균락PCR결과시음성전화자득도198 bp편단,양성전화자득도333 bp편단；측서결과여예기상부합；Real-time PCR시소득병독적도약위2×109TU:Western blot시Tβ4전염조재33 KD처유특정조대.결론 성공구건대서Tβ4기인만병독재체pGC-FU- Tβ4,병건립만병독과표체계통.
Objective To construct the recombinant lentiviral vector over-expressing the rat thymosin beta 4 (Tβ4) gene,to observe its expression in 293T cells,and to provide an experimental foundation for the treatment of myoeardial infarction at the gene level.Methods The rat Tβ4 gene was obtained from genbank,and connected into the linearized lentiviral vector to generate the lentiviral over-expressing vector named pGC-FU-Tβ4.The positive clones identified by polymerase chain reaction (PCR) were sequenced after they was transformed into bacterial competent cells.The concentrated supernatants were collected for titer measurement by using Real-time PCR after pGC-FU-Tβ4,pHelperl.0,and pHelper2.0were eo-transfected into 293T cells.The Tβ4 was detected by using Western blotting after transfecting the 293T cells with the acquired recombinant lentivirus.Results A target gene fragment of 143 bp was obtained by enzyme digestion.The negative transformants of 198 bp and the positive transformants of 333 bp were obtained by PCR.The sequeneing results were consistent with the expectation.The titer of comcentrated lentivirus reached 2 × 109 TU/ml.A characteristic band of 33 KD could be found.Conclusion The recombinant lentiviral vector of the rat Tβ4 was successfully constructed and packaged,which laid the foundation for further application of the Tβ4 to the treatment of myocardial infarction at the gene level.