Breeding Of Mammary Gland-specific Transgenic Rabbits Expressing RhPA And Purification And Analysis Of The Expressed Products

Human tissue plasminogen activator(t-PA)has the problems of short half-life,easy inactivation,and risk of internal hemorrhage.The recombinant human tissue plasminogen activator(rhPA),which we have adapted accordingly,has the advantages of prolonged half-life,increased yield,and highly compatibility with human body.And most of the existing recombinant proteins were expressed in prokaryotic E.coli,and a few were expressed by animal or insect cells.Separation and purification of rhPA from milk has not been reported.Therefore,in this study,we used a combination of several purification methods to ensure the purity,the thrombolytic activity and the stability of the recombinant human plasminogen activator(rhPA)purified for transgenic rabbits milk.Recombinant human plasminogen activator(rhPA)transgenic rabbits were screened and bred.A goat BLG signal peptide coding region and the coding sequence of the K2 and P domains of human t-PA was cloned into PCL25 which contained a 5’ genomic fragment of goat β-casein gene and a human cytomegalovirus immediate-early promoter/enhancer serves as a chimeric promoter/enhancer.The constructed mammary gland-specific expression vector was named PCL25/rhPA.After prokaryotic microinjection of the linear fragments of the vector,a total of 48 surviving puppies were obtained and 27 positive rabbits were detected by PCR.The whey samples of the FO transgenic rabbits were detected by ELISA,results showed that 11 transgenic rabbits expressed rhPA in the milk.4 homozygous transgenic rabbits were obtained by the copulation of the F2 transgenic rabbits.The homozygous and hemizygous rabbits whey samples were detected by ELISA.It was found that the expression level of rhPA in the transgenic homozygous rabbits whey was significantly higher than hemizygous transgenic rabbits.The thrombolytic activity of the diluted rabbit whey and alteplase were measured by FAPA,results showed that both homozygous and hemizygous rabbits whey expressed obvious thrombolytic activity and produced transparent circle on the fibrin plate.But homozygous transgenic rabbits showed stronger thrombolytic activity than hemizygous transgenic rabbits.SDS-PAGE electrophoresis and Western Blot analysis of the transgenic rabbit whey revealed that the rhPA-containing rabbits whey had a band around 41 KDa,which matched the theoretical size of the rhPA.Purification and analysis of rhPA purified from transgenic rabbit milk.In this experiment,milk samples were pretreated by 20000 g centrifugation,35%saturated ammonium sulfate precipitation,acid-base precipitation and ultrafiltration.Insoluble substances such as casein,fat and most of salt ions and small molecule substances were removed.And the affinity,ion exchange,and gel filtration chromatography were used to further purify rhPA.It was found that most of the target proteins could be eluted by Lysine HyperD affinity chromatography using 0.5 mol/L L-Arginine,0.5 mol/L NaCl in 25 mmol/L PB buffer solution(pH 5.5).When affinity chromatography was performed on Blue Sepharose 6FF,washed with 0.4 mol/L NaCl in 25 mmol/L PB buffer solution(pH8.0)and then eluted with 1 mol/L NaCl in 25 mmol/L PB buffer solution(pH8.0)was better.The optimal exchange ion concentration was searched by ion exchange chromatography and it was found that the separation effect was better when washed with 0.3 mol/L NaCl in 20 mmol/L PB buffer solution(pH 5.0)and then eluted with 1 mol/L NaCl in 20 mmol/L PB buffer solution(pH 5.0).Finally,the target protein rhPA was further purified by using gel filtration chromatography of Surpdex G75.The elution products of each chromatographic were biologically active by the FAPA and showed a band around 41 KDa by SDS-PAGE at the same time.The purified rhPA by various of purification was semi-quantitatively compared to the original whey by ELISA,the recovery rate was 30%.The Quantity One 1-D software was used for SDS-PAGE purity analysis and the purity was more than 96%.Western Blot showed that the purified product contained a band around 41 KDa,which was consistent with the size of the rhPA target protein.The thrombolytic bioactivity of the final purified rhPA was compared to alteplase by the FAPA method.It was found that the purified product maintains a highly thrombolytic activity in vitro and the activity is about 150 times than that of alteplase.The molecular weight was 41713 Da determined by time-of-flight mass spectrometry.N-and C-terminal protein sequence of rhPA further indicated that the purified protein is intact.These results confirmed that the purified product was a recombinant human plasminogen activator.In this study,transgenic rabbit which highly express rhPA in the milk was screened,and the expression level and in vitro thrombolytic activity of the rhPA homozygous transgenic rabbits were higher than that of the hemizygote transgenic ones.Using a combination of multiple purification strategies,the purified rhPA was obtained with a specific activity about 150 times than that of the alteplase by FAPA.It provides new insights for the subsequent purification studies.