Construction Of Expression Vectors For Human Interferonα-2b Gene In Both Nucleus And Chlorplast Of Tobacco And The Transformation

Interferons have many clinical applications due to their abilities of inhibiting viral replication,cell growth and enhancing the immune response. Human interferonα-2b (HuIFNα-2b) ranks third in world market use for biopharmaceutical. However, HuIFNα-2b is unavailable to most patients in developing countries because of its high cost. In recent years, plant based expression system has become the hot topic of research for producing biopharmaceutical proteins for prevention or therapy in the large scale. Comparing to some traditional expression systems such as microbial system and animal cells, transgenic plants have many advantages. The most attractive advantage is the comparatively low cost. Besides, transgenic plants, which belong to eukaryotic organisms can produce biopharmaceutical proteins with fully biological activity. Moreover, human can avoid the comtamination from pathogenic, oncogenic DNA sequences and endotoxins of plants. In the present research, we constructed HuIFNα-2b expression vectors both for nucleus and chloroplasts intergration in tobacco,and successfully transformed the vector for nucleus into tobacco nucleus genome via Agrobacterium-mediated method. The main results are as follows..1)The prokaryotic expression vector pet28a-IFN for HuIFNα-2b was constructed and transformed into E.coli. The expression of HuIFNα-2b was comfirmed by SDS-PAGE.2)The nuclear expression vector pBIFN for HuIFNα-2b was constructed and transformed into tobacco leaf discs via Agrobacterium-mediated method. Thirty five positive regenerated transformants were obtained .3 ) The site-specific transformation and expression vector pCL151-IFN for HuIFNα-2b in tobacco chloroplasts was constructed. HuIFNα-2b gene,which contains the transcription promoter of Prrn gene and the 3'transcription terminator of psbA gene, was inserted between two homologous fragments(rbcL and accD) of tobacco chloroplast genome to form pCL151-IFN.In conclusion, the prokaryotic expression for HuIFNα-2b gene, nuclear transformation for HuIFNα-2b gene in tobacoo and the construction of cloroplast transformation and expression vector in tobacco are completed. The achievements of present research has paved the way for HuIFNα-2b production in transgenic tobacoo, especially in chloroplasts.