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目的:构建内皮抑素-可溶性血管内皮细胞生长抑制因子融合基因重组腺病毒载体并包装成重组腺病毒,为下一步基因治疗打下基础。方法:用PCR方法在hENDO基因的5’端引入IL_3信号肽、3’端引入弹性蛋白连接肽序列(linker),再与sVEGI基因连接,插入到pCAl3腺病毒载体中,用脂质体介导包装出重组腺病毒,PCR法对重组腺病毒进行鉴定。结果:构建完成hENDO-sVEGI融合基因重组腺病毒载体,融合基因包括IL_3信号肽、hENDO全长基因、弹性蛋白连接肽序列和sVEGI基因,总长度约1114bp,经酶切鉴定、序列分析证实克隆序列、插入方向和读码框架均正确,可包装出携带融合基因的重组腺病毒,用TCID_(50)法测定粗制重组腺病毒滴度为2×10~(11) TCID_(50)/L。结论:成功构建了hENDO-sVEGI融合基因,连接肽序列使两蛋白空间构象不受影响,IL_3信号肽保证蛋白可被分泌至胞外,完成携带融合基因的重组腺病毒的包装与鉴定,为进一步开展肿瘤基因治疗研究奠定了基础。
OBJECTIVE: To construct endostatin-soluble vascular endothelial growth factor (VEGF) fusion gene recombinant adenovirus vector and package it into recombinant adenovirus, which lays a foundation for further gene therapy. METHODS: The IL 3 signal peptide was introduced into the 5′ end of the hENDO gene by PCR, and the 3′ end was introduced into the elastin linker (linker). The sVEGI gene was then ligated into the pCAl3 adenovirus vector and mediated by liposome. The recombinant adenovirus was packaged and the recombinant adenovirus was identified by PCR. RESULTS: The hENDO-sVEGI fusion gene recombinant adenovirus vector was constructed. The fusion genes included IL 3 signal peptide, hENDO full-length gene, elastin linker peptide sequence and sVEGI gene. The total length was about 1114bp. The cloned sequence was confirmed by restriction analysis and sequence analysis. The insertion direction and reading frame were correct, and the recombinant adenovirus carrying the fusion gene could be packaged. The titer of the recombinant adenovirus was determined to be 2×10 11 TCID_(50)/L by the TCID 50 method. Conclusion: The hENDO-sVEGI fusion gene was successfully constructed, and the peptide sequence was connected so that the spatial conformation of the two proteins was not affected. The IL-3 signal peptide ensured that the protein could be secreted to the outside of the cell and the packaging and identification of the recombinant adenovirus carrying the fusion gene was completed. It lays the foundation for carrying out tumor gene therapy research.