Expression Of Recombinant Human Serum Albumin In Transgenic Alfalfa And Ryegrass

Human serum albumin(HSA),the most abundant protein in human plasma,plays an important role in maintaining plasma oncotic pressure,anticoagulate and free radicals scavenging.This protein is widely used in the clinic to treat hypoproteinaemia,fluid loss due to burn injuries,nephrotic syndrome,liver cirrhosis,pulmonary encephalopathy,skin eruptions and so on.Due to the large dose of HSA treatment and insufficient blood source,the output can not meet the market demand.The current rHSA expression system still does not solve the problem of shortage of HSA sources.In this regard,we explored the expression pattern of recombinant HSA,optimized the tissue culture and plant regeneration conditions of alfalfa(Medicago Sativa L.)and ryegrass(Lolium perenne L.),and established their genetic transformation systems.The HSA gene was introduced into alfalfa and ryegrass respectively by Agrobacterium-mediated transformation.Through the identification of genomic PCR and Western blotting,transgenic alfalfa lines and transgenic ryegrass lines expressing rHSA were obtained.It laid the foundation for the subsequent isolation and purification of HSA,physicochemical properties and identification of pharmacological properties.The main experimental results obtained in this study are as follows:1.A plant expression vector was constructed based on the basic sequence of human serum albumin and transferred to Agrobacterium strain GV3101 by electroporation.After PCR identification,positive transgenic engineering bacteria were obtained.2.The genetic transformation system was established using cotyledons of the alfalfa cultivars "Eureka" and "Sardi" as explants.The experimental results obtained are as follows:(1)The optimum medium for the induction of callus and embryogenic callus of alfalfa is MS medium + 2 mg/L 2,4-D + 0.2 mg/L KT + 2 g/L casamino acid,3%sucrose,pH 5.8.(2)The optimal medium for differentiation induction is MS medium + 0.5 mg/L KT?2 g/L casamino acid,3%sucrose,pH 5.8.(3)The rooting medium is 1/2 MS medium,1.5%sucrose,pH 5.8.3.The genetic transformation system was established using seed embryo of the ryegrass cultivar "Tetragold" as explants.The experimental results obtained are as follows:(1)The optimum medium for the induction of callus is MS medium + 5 mg/L 2,4-D +0.2 mg/L 6-BA + 2 g/L casamino acid,3%sucrose,pH 5.8.(2)The optimum medium for the induction of embryogenic callus of ryegrass is MS medium + 3 mg/L 2,4-D + 0.1 mg/L 6-BA + 2 g/L casamino acid,3%sucrose,pH 5.8.(3)The optimal medium for differentiation induction is MS medium + 2 mg/L 6-BA +0.1 mg/L NAA + 2 g/L casamino acid,3%sucrose,pH 5.8.(4)The rooting medium is 1/2 MS medium+ 0.1 mg/L IBA,1.5%sucrose,pH 5.8.4.A gradient experiment was performed on the concentration of the bacteria and the time of the infusion in the process of Agrobacterium transformation.The results showed that the optimum condition for the transformation of alfalfa was OD600 = 0.6,and the duration of infection was 10 min.And the optimal condition for the transformation of ryegrass was OD600 = 0.6,and the immersion was 5 min.5.Agrobacterium-mediated transformation was used to transform callus of alfalfa and ryegrass respectively.Plant regeneration was induced and positive transformed plants were screened.By genomic PCR and Western blotting detection,it was finally determined that the HSA gene was successfully transferred into transgenic lines of alfalfa and ryegrass,respectively.According to statistics,96 strains were obtained for transgenic "Sardi",165 strains for transgenic "Eureka",and 119 strains for transgenic "Tetragold".