TRADD基因外显子的拼接及重组腺病毒的构建

The Splicing of Exons of TRADD Gene and the Construction of TRADD Recombined Adenovirus

目的构建含有人TRADD基因片段的重组腺病毒载体。方法从人肝组织中提取总RNA,逆转录得到cDNA。按照TRADD基因4个外显子的基因序列设计引物,分别扩增得到4个外显子的基因片段。对4个外显子进行基因拼接,得到全长的TRADD片段。利用AdEasy系统构建TRADD重组腺病毒,测定病毒滴度,并转染成纤维细胞。结果拼接成功939kb的TRADD基因,行PCR扩增重组腺病毒中TRADD基因,在略小于1000kb处见到明显的TRADD条带。结论构建成功的含有TRADD基因片段的重组腺病毒,可用于转染人成纤维细胞,用于检测TRADD对病理性瘢痕成纤维细胞的影响。

Objective To construct recombinant adenovirus vector carrying human TRADD gene. Methods The total RNA was extracted from human liver, then it was used to amplify cDNA by RT-PCR. Primers were designed according to the four exons of TRADD gene, They were used to amplify four exons of TRADD. Then these four exons were linked to form the TRADD cDNA. The latter was used to construct adenovirus vector by the AdEasy adenovirus construction system. The titer of the adenovirus was examined. Results The 939 kb TRADD gene was spliced. It was examined by PCR amplification. Conclusion The recombinant adenovirus vector carrying human TRADD gene was successfully constructed. It can be used for detecting the effects of TRADD on fibroblasts of hypertrophic scar and keloid.