Expression And Biological Activity Identification Of Human Pigment Epithelium-derived Factor

PARTⅠExpression and Biological Activity Identification of Human Pigment Epithelium-derived Factor in E.coliBackground Retinopathy of prematurity(ROP) is the major cause of blindness in children in the world. Medical intervention is key point of the present research. The inhibition of angiogenesis would be an effective strategy to treat ROP which abnormal angiogenesis underlies the main pathology. Human pigment epithelium derived factor (PEDF) is the most potent natural inhibitor of angiogenesis. PEDF can effectively inhibit the retinal neovasculization in ROP animal models, so we try to find a simple and efficient method to recombinate the PEDF.Objective To express and purify the recombinant human pigment epithelium-derived factor (rhPEDF) which inhibits the proliferation of the endothelium cells from blood vessel in Escherichia. coli, and which is a well known expression system as a simple, convenient, and economic way to obtain this protein with intact biological activity.Methods The up and down primers contains EcoRⅠand HindⅢsites respectively were designed based on the mature peptide sequence of human PEDF cDNA in the GeneBank. The PCR product was digested with EcoRⅠand HindⅢ, and the 1.2 kb fragment was recovered by agarose electrophoresis. The vector was digested with EcoRⅠand HindⅢ, and the 5.3 kb fragment was recovered by agarose electrophoresis too. The 1.2 kb human PEDF DNA fragment and 5.3 kb pET28a fragment were ligated by DNA ligation kit at 16℃overnight and transformed into E.coli DH5a. The transformed bacteria were spread onto the culture dish with Kana and cultured in an incubator at 37℃. The recombinant plasmid was extracted and identified by EcoRⅠHindⅢdigestion, PCR amplification and DNA sequencing. Then recombinant plasmid was transformed into E.coli BL21(DE3) to establish the engineering strain for expression of PEDF. After purification by His-tag affinity chromatography, the recombinant protein refolds to its natural structure by dialysis in the presence of DTT. The biological activity of the recombinant human PEDF protein was determined by human umbilical vein endothlial cell (HUVEC) survival assay with the MTT viability measurement. Results PCR amplification, restrictive enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the prokaryotic expression vector pET28a, and expressed mainly in the form of inclusion bodies after IPTG induction. The recombinant protein was 50kDa of molecular weight and mainly in the form of inclusion bodies detected by Western blot analysis. The insoluble rhPEDF was solublized from inclusion bodies by denaturation in 8M urea and renatured by dialysis in the presence of DTT. An average of 8.3 mg of purified protein was obtained from 1 L of culture. It significantly inhibited the proliferation of the HUVEC line in a concentration-dependent manner. It showed an EC50 of approximate 80 nmol/L.Conclusions The engineering strain pET28a/PEDF-BL21(DE3) was constructed, which could express the mature peptide of human PEDF. A method for collecting soluble rhPEDF with biological activities from inclusion bodies by denaturation and renaturation was developed. This protein could be a potential medication in preventing and managing retinopathy of prematurity.PARTⅡTransient Expression and Biological Activity Identification of Human Pigment Epithelium-Derived Factor in Mammary Cell Line SP2/0Objective The human PEDF cDNA was inserted into eukaryotic expression vector pIRESneo3 to construct expression vector pIRESneo3-PEDF. The successfully constructed pIRESneo3-PEDF expression vector was transfected into SP2/0 cells mediated by LipofectamineTM 2000. The transient expression in SP2/0 cells was identified by Western blot.Methods The up and down primers contains EcoRⅠand NotⅠsites respectively were designed based on the mature peptide sequence of human PEDF cDNA in the GeneBank. The PCR product was digested with EcoRⅠand NotⅠ, and the 1.2 kb fragment was recovered by agarose electrophoresis. The vector was digested with EcoRⅠand NotⅠ, and the 5.2 kb fragment was recovered by agarose electrophoresis too. The 1.2 kb human PEDF DNA fragment and 5.2 kb pIRESneo3 fragment were ligated by DNA ligation kit at 16℃overnight and transformed into E.coli DH5a. The transformed bacteria were spread onto the culture dish with Amp and cultured in an incubator at 37℃. The recombinant plasmid was extracted and identified by EcoR I Not I digestion, PCR amplification and DNA sequencing. The expression vector pIRESneo3-PEDF and LipofectamineTM 2000 were mixed and transfected into SP2/0. After cultured for 24h, the recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot. The biological activity of the recombinant human PEDF was measured by the method of MTT.Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. Western blot analysis showed that cell culture supernatant could react with McAbs of mouse anti-human PEDF. There is only one obvious band at the position of relative molecular weight 50 KDa, and it is equivalent to the expected value. It significantly inhibited the proliferation of the human umbilical vein cell line HUVEC.Conclusions The expression vector of pIRESneo3-PEDF has been fulfilled and transiently expressed successfully, which laid a foundation for futher study of stable expression and purification. This protein could be a potential medication in preventing and managing retinopathy of prematurity.