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目的:通过构建人干扰素α-2b(hIFNα-2b)基因的植物表达载体,为后期将其导入胡萝卜愈伤组织中作准备。方法:采用PCR技术从人基因组DNA扩增hIFNα-2b编码基因及全长基因,将其克隆于pMD19-T载体中,双酶切hIFNα-2b基因及植物表达载体pBI121,回收目的片段,T4DNA连接酶连接得到植物表达载体pBI121-hIFN。采用三亲交配法将后者导入根瘤农杆菌。结果:经PCR检测,双酶切及DNA序列测定表明hIFNα-2b编码基因及全长基因已分别插入植物表达载体pBI121中,重组表达载体pBI121-IFN已成功转化根瘤农杆菌LBA 4404。结论:成功构建了植物表达载体pBI121-hIFN并转入根瘤农杆菌。
OBJECTIVE: To construct a plant expression vector for human interferon alpha-2b (hIFNα-2b) gene in preparation for its introduction into carrot callus in the later stage. Methods: The hIFNα-2b gene and its full-length gene were amplified by PCR from human genomic DNA, cloned into pMD19-T vector and double-digested with hIFNα-2b gene and plant expression vector pBI121. The target fragment was recovered and T4 DNA was ligated The enzyme was ligated to obtain the plant expression vector pBI121-hIFN. The latter is introduced into Agrobacterium tumefaciens by the method of mating with three parents. Results: The results of double enzyme digestion and DNA sequencing showed that the encoding gene of hIFNα-2b and the full-length gene were inserted into the plant expression vector pBI121 respectively. The recombinant vector pBI121-IFN was successfully transformed into Agrobacterium tumefaciens LBA 4404. Conclusion: The plant expression vector pBI121-hIFN was successfully constructed and transformed into Agrobacterium tumefaciens.