The Expression And Purification Of HBRP In The Prokaryotic Cell

INTRODUCTIONBovine seminal plasma protein family secreted by the seminal vesicles. Bovine seminal plasma is the major component of bovine seminal plasma. The major function of the bovine seminal plasma protein is to accelerating the capacities of bovine epididymal sperm. The bovine seminal plasma proteins bind to epidid-ymal spermatozoa via their interaction with choline phospholipids after ejaculation. In addition, they interact with bovine sperm capacitating factors, heparin and HDL. Bovine seminal plasma proteins may stimulate cholesterol and phospholipids efflux from the sperm membrane, which is the important step of the ca-pacitaion.Clone a new cDNA gene sequence related to BSP, and make it expressed in eukaratic cells. HBRP ( Human BSP - Related Protein) is homologous to BSP proteins. The fusion protein acquired is purified using affinity chromatography. So what we have done is of great significance to study further the biological functions of the new cloned protein related BSP proteins.MATERIALS AND METHODSMaterials1 Plasmid and bacteria1) pGEX expression vector pGEX -5X - 1 (Pharmacia)2) E. coli BL21(Pharmacia) Reagents1) DNA Fragment Recovery Kit (Beijing Dingguo Company)2) DNA Marker (TaKaRa)3) Restriction enzyme BamHI, XlioI(MBI)4) T4DNA Ligase (TaKaRa)5) Prepacked Glutathione Sepharose 4B (Pharmacia)6) IPTG(TaKaRa)7) GSH( Sigma)8)1- chilro -2,4- dinitrobenzene( CDNB ) ( Sigma) 9 ) anti - GST Rabbit polyclonal antibody 10) mouse - anti - rabbit second antibody Methods1 Cloning the gene into a pGEX expression vectorClone the HBRP gene using PCR, restriction digest the PGEX - 5X - 1 vectors and PCR product, recovery the PCR product and PGEX - 5X - 1 vectors . ligate the insert HBRP to a pGEX - 5X -1 expression vector. Prepare Eoli. BL21 competent cells. Transform PGEX - HBRP into the Eoli. BL21 competent cells. Isolate Small - scale PGEX - HBRP. Screening using restriction enzyme . Screening using PCR . Sequencing of pGEX - HBRP2 Inducing and expression of pGEX - HBRP fusion protein1) Optimizing the inducing conditionsChange the concentration of IPTG and the inducing temperature, choose the best inducing conditions. Inducing expression. Get the inclusion bodies2) Western blot analysis of fusion protein expression(1) Electrophoresis (2) transfer proteins from gel to membrane (3) blocking (4) incubation with primary antibody (5) enzyme conjugate incubation (6) substrate incubation.3 ) GST Detection Module with CDNB enzymatic assay 3 Purification of GST fusion proteins1 The denaturalization of inclusion bodies.2 Purification using Glutathione Sepharose 4B columnWash matrix with 1 PBS, prepare a 50% slurry for batch purification method , pack column with matrix slurry. Add the sample to prepared affinity matrix, wash bound matrix with 1 PBS, elute fusion proteins using glutathione elution buffer.RESULTS1. Cloning the gene into a pGEX expression vectorClone the HBRP gene using PCR, restriction digest the PGEX - 5X - 1 vectors and PCR product with BamH I and Xho I , recovery the PCR product and PGEX - 5X - 1 vectors . Clone the gene HBRP into a pGEX - 5X - 1 expression vector. Transform PGEX - HBRP into the Eoli. BL21 competent cells. Select the reactive clone in the Amp plate.2. The ORF of the HBRP coding a peptide of 233 aa, using the DNASIS v2. 5 Demo analysis its structure and PI3. Screening1) Screening using restriction enzyme.The plasmid was tested by the restriction enzyme BamH I and Xho I incision, and 1% agarose gel electrophoresis. The results show that there were two bands at the respected sites about 4.9kb and 750bp respectively. It means that HBRP has been cloned into a pGEX -5X - 1 expression vector correctly.2 ) Screening using PCR . Sequencing of pGEX - HBRPThe plasmid was screened by PCR, and 1% agarose gel electrophoresis. The results show that there was one band at the respected sites about 750bp. Suggested that there is HBRP gene in the plasmid.4. Sequenc...