Expression Of Human Lactoferrin,Thymosin α1 Fusion Gene In The Yeast Pichia Pastoris And Lettuce

Human Lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. It is present in milk, tear, saliva and other secretions of mammals, the length of open read frame of human lactoferrin gene is 2.14kb and the protein molecular weight is 80kD. Thymosin alpha 1 (Tα1) is a polypeptide hormone that present in all tissue and organ of mammals. Tα1 is a highly conserved 28 amino acid peptide, its molecular weight is 3.108kD and pI is 4.2. With the more and more important biological function of these two proteins had been found ,such as body immunomodulatory,anticancer and inhibition virus etc, the studies about human lactoferrin and Tα1 had became a hot point all over the world. In this study, we synthesized hLf gene and fused with Tα1 gene, then expressed the fusion gene Tα1hLf in the Pichia Pastoris and lettuce. To enhanced the expression amount of Tα1hLf in the Pichia Pastoris and lettuce, we applied another gene, Vitreoscilla hemoglobin gene (vgb), as assistant gene. In the synthesis experiment of fusion gene: According to the published cDNA of hLf and considering the enzyme identified sites, designed and synthesized 51 single chain of nucleotide. Produced 7 gene fragments and 2 linker, then connected these fragments into whole hLf and fused with Tα1 gene(100bp). Sequence analysis result shows that the sequence and read frame of fusion gene are corrected. In the fusion gene expression experiment on Pichia Pastoris: Used pPIC9K to construct yeast expression vector pPIC9K-vgbTα1hLf which can intracellular expression of vgb and extracellular secreted expression of hLf. After transgenic pPIC9K-vgbTα1hLf into yeast, through experiment of PCR and SDS-PAGE verified that fusion gene had combined on yeast genome and expressed fusion protein about 83 kD. Western blotting and inhibit bacterium experiments indicated that human lactoferrin of fusion protein has natural activity, thus verified the exactness of hLf and fusion gene. Difference spectral analysis and hypoxic experiment indicate VHb have activity and can promoting yeast growth and fusion gene expression. Shake flask cultivation experiment shows that the expression amount of fusion protein are 30mg/L. In the fusion gene expression experiment on lettuce: Used pGΩ4A to construct two middle vectors that started by RuBP promoter and 35S promoter respectively. Then linked the fusion gene express box with VHb gene express box and cloned on pBI121, which appeared plant express vector pGBIVHbTα1hLf (vgb and fusion gene started by 35S promoter) and pGBIRVHbTα1hLf (vgb started by 35S promoter, fusion gene started by RuBP promoter). Utilized agrobacterium to transform these two plant expression vectors into lettuce and obtained 46 individual regeneration seedling, which measured by PCR and ELISA, the results indicated that fusion protein had expressed, difference spectral analysis also indicated that vgb gene had expressed and taken on activity.