大鼠胸腺素β4基因重组慢病毒载体的构建及体外表达

Construction and expression in vitro of the recombinant lentiviral vector containing the rat thymosin beta 4 gene

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Tp4的表达。方法从Genbank获取大鼠TB4基因序列，化学合成后连接人线性化慢病毒载体pGC—FU，得到重组慢病毒载体pGC—FU—T[34，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—T134、pHeIper1．0、pHelper2．0共转293T细胞，收集上清液浓缩后使用实时定量PCR（Real—timePCR）进行滴度检测。用所得病毒感染293T细胞后，Westernblot检测TB4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143bp；菌落PCR结果示阴性转化子得到198bp片段，阳性转化子得到333bp片段；测序结果与预期相符合；Real—timePCR示所得病毒滴度约为2×109TU；Westernblot示T134转染组在33KD处有特征条带。结论成功构建大鼠T134基凶慢病毒载体pGC—FU—T[34，并建立慢病毒过表达系统：

Objective To construct the recombinant lentiviral vector over-expressing the rat thy- mosin beta 4 （Tβ4） gene, to observe its expression in 293T cells, and to provide an experimental founda- tion for the treatment of myocardial 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 se- quenced after they was transformed into bacterial competent ceils. The concentrated supernatants were col- lected for titer measurement by using Real-time PCR after pGC-FU-Tβ4, pHeIperl. 0, and pHelper2.0 were co-transfected into 293T cells. The Tβ4 was detected by using Western blotting after transfecting the 293T cells with the acquired reeombinant lentivirus. Results A target gene fragment of 143 bp was ob- tained by enzyme digestion. The negative transformants of 198 bp and the positive transformants of 333 bp were obtained by PCR. The sequencing results were consistent with the expectation. The titer of concentrat- ed lentivirus reached 2 × 109 TU/ml. A characteristic band of 33 KD could be found. Conclusion The re- combinant lentiviral vector of the rat Tβ4 was successfully constructed and packaged, which laid the foun- dation for further application of the Tβ4 to the treatment of myocardial infarction at the gene level.