Specific Expression Of Recombinant RhPA In Goat Mammary Gland And Product Isolation And Analysis

At present,thrombolytic drugs including urokinase,streptokinase and human tissue type plasminogen activator were used in clinic treatment.Plasminogen activator has developed to the 3th generation,and the recombinant human plasminogen activator represents the latest of them.The F,E and K1 deletant of human plasminogen activator(rhPA)has the advantages of strong activity of thrombolysis,high specificity of fibrin affinity,prolonged half-life,reduced dosage,and reduced incidence of general bleeding and intracranial hemorrhage.Using mammary gland bioreactor to produce rhPA greatly improves the production capacity and reduces the cost.Protein can be translated,phosphorylated,carboxylated and other modificated in vivo,and has high biological activity therefore.The recombination of hyperfiltration and affinity chromatography could obtain rhPA from milk effectively.In this experiment,rhPA transgenic goats were prepared by somatic cell nuclear transfer(SCNT),and the transgene rhPA was expressed in the mammary gland of goats specifically.Results of ELISA and Western Blot shew that rhPA was secreted into the goat milk,while FAPA indicated that the recombinant protein has a high specific activity.Goat fetal fibroblasts were derived from 30 day old fetus by sterile surgery.The mammary gland specific expression vector BCL14/rhPA was transfected into fetal fibroblast cells by electroporation.Resistant clones appear after been screened by selective medium containing G418(600 ?g/mL)for about 7?10 d.The monoclonal cells lines were picked up.PCR analysis indicated that 24 of the resistant clones were transgenic cell lines,and 7 lines were selected as the donor cells.The positive cells were hungered for 72h with DMEM/F12 medium containing 0.5%FBS and then were used as donor cells.Metaphase ?(M ?)oocytes were collected from oviducts of donor goats as recipient cytoplasts,reconstructed embryos were transplanted into the oviducts of the receptors.Pregnancy was determined by abdominal ultrasound examination 30 days and 60 days after the SCNT,and PCR analysis was conducted to detect the integration of the rhPA gene.256 reconstructed oocytes were transferred into 26 forster mother,and 7 of them were pregnant.The pregnancy rate was 26.9%.We got 2 lambs and they were named BP21 and BP22 respectively,results of PCR analysis shew that both of them were transgenic ones.The cloned goats were mated with normal rams after mature.The lambs of them were analyzed by PCR and results shew they were transgenic too.Milk were collected and high-speed centrifuged to get whey.SDS-PAGE and Western Blot showed a 39KDa specific band.The expression level of rhPA in transgenic goat whey was about 92?g/mL by ELISA.Results of ELISA and the in vitro thrombolysis test FAPA shew that specific activity of the rhPA in the whey was 40 times stronger than commercial alteplase.In this experiment,rhPA transgenic goats were prepared by somatic cell nuclear transfer(SCNT),and the exogenous gene rhPA expresses in the mammary gland specifically.Results of ELISA and Western Blot shew that rhPA was secreted into the milk,while FAPA indicated that the recombinant protein has a high enzyme specific activity.The purification and authentication of rhPA:the milk were high speed centrifuged and precipitated by HCl and NaOH to get whey,and then a lysine affinity chromatography was conducted.SDS-PAGE and Western Blot shew a 39 kDa specific objective band,and result of FAPA indicated that rhPA in milk kept its biological activity after lysine affinity chromatography.In this experiment,rhPA transgenic goats were produced by SCNT,rhPA in the milk had a high specific activity,and the concentration was about 73.6?g/mL.The recombinant protein rhPA could be purified by lysine affinity chromatography with a high efficiency while kept it's high biological activity,it laid a foundation for further purification experiment.There's no report about rhPA expressed by goat mammary gland at present.