Vasostatin重组腺病毒的构建和体外表达的研究

目的构建vasostatin重组腺病毒表达载体，并进行体外表达研究。方法根据GenBank中提供的钙网蛋白序列，设计并合成引物，以人骨骼肌cDNA文库为模板用PCR扩增出人vasostatin基因，将其克隆入T载体。经酶切及测序鉴定后亚克隆至穿梭载体，与经过限制酶处理的骨架腺病毒载体DNA—TPC复合物共转染293细胞。用Western blot检测体外表达。结果PCR扩增出约590bp产物，测序结果与文献报道一致。用PCR及Western blot证实重组腺病毒构建成功，能有效的表达出vasostatin蛋白。结论vasostatin重组腺病毒载体能有效的在体外表达出vasostatin蛋白。

Construction of Recombinant Adenovirus for the Angiogenic Inhibitor,Vasostatin,and Its Expression in vitro

Objective To construct a recombinant replication deficient adenovirus for the angiogenic inhibitor, vasostatin and assay its expression in vitro. Methods The cDNA for vasostatin was got by PCR amplification, then it was cloned into T vector and confirmed by enzymatic analysis and direct sequencing. Subsequently the cDNA was subcloned into the shuttle plasmid, and co transformed into 293 cells with backbone plasmid. The resulting recombinant andenovirus was confirmed by PCR and western blot. The activity was assayed by inhibition of HUVEC growth. Results The PCR product was about 590 bp in length, and sequencing result was identical to that reported. The construction of recombinant replication deficient adenovirus was confirmed by PCR. Western blot analysis showed the expression of vasostatin in vitro. The supernatant from transduced HeLa cells inhibited the growth of HUVEC specifically. Conclusion The recombinant adenovirus for vasostatin efficiently mediated the expression of the protein in vitro.