Agrobacterium-mediated Transformation And Expression Of Human Serum Albumin Gene In Physcomitrella Patens

Human serum albumin(HSA)is a protein in human plasma,and its non-glycosylated single-chain polypeptide contains 585 amino acids with a molecular weight of 66 kD.Clinically,HSA can be used to treat shock and burns.It can be used to supplement blood loss caused by surgery,accidents,or major bleeding.It can also be used as a plasma compatibilizer.It plays an important role in clinical and biotechnology,and there is a huge market demand.Simply extracting HSA from human blood is difficult to meet the market demand.Moreover,human blood sources can easily lead to various pollutions and the spread of diseases.In order to get rid of the limited production capacity of plasma sources and to address the risk of virus transmission using commercially available HSA,it is an important application field for plant bioreactors to use plants to produce pharmaceutical proteins such as antibodies or therapeutic proteins.Physcomitrella patens has great advantages in biotechnology as a heterologous expression host for recombinant therapeutic proteins and commercially valuable natural products.Because of its short life cycle and easy culture,transgenic plants are easy to analyze.Moreover,Physcomitrella patens is the most highly recombined plant in terrestrial plants and is called "green yeast." The nuclear genome is easy to homologously recombine with the exogenous DNA with homologous fragments.The clonal growth of Physcomitrella patens in haploid form makes the level of genetic variation relatively low.These advantages facilitate genetic transformation and screening after gene manipulation.The whole genome sequence of Physcomitrella patens has been completed,which enables its bioinformatics research and functional genome comparison to be performed.Therefore,Physcomitrella patens was selected as a bioreactor to produce recombinant HSA.The use of Agrobacterium tumefaciens-mediated transfer of exogenous genes into Physcomitrella patens has not been reported in China.The specific research content and results are as follows:1.The pYB1906 vector was modified and the rHSA gene was synthesized using the PTDS gene synthesis method and ligated into the pYB1906 vector to construct a new plant vector pYB2924.This vector introduced the Physcomitrella patens MTH1 protein secretion signal peptide sequence at the 5?end of the HSA gene.To facilitate the realization of secretory expression in Physcomitrella patens.2.The Agrobacterium tumefaciens transformation system was used.The pYB2924 gene was introduced into the Physcomitrella patens by infecting protoplasts with Agrobacterium.Obtained positive transformed plants with hygromycin resistance.And successfully cultured stably transformed strain is capable of rHSA secreted,mature established Agrobacterium-mediated system.3.The positive transgenic plants screened by PCR amplification and RT-PCR detection confirmed that the pYB2924 gene was integrated into the genome of Physcomitrella patens and was expressed at the transcriptional level.The expression of the protein was confirmed by SDS-PAGE electrophoresis.Finally,ELISA test showed that rHSA was secreted in Physcomitrella patens and the expression level was27 mg/L according to the ELISA result.