Cloning Of Full-Length Cdna,Prokaryotic Expression And Monoclonal Antibody Preparation Of Sheep Cell Division Cycle42

Brucellosis was a zoonotic pathogens disease with a highly contagious caused by Brucella. As it’s outbreaks and harmful to human health and economic development of animal breeding industry in China. Take an effectiveprevention and treatment of brucellosis have been imperative. In recent years, the cell surface antigen molecules targeting treatment has become a very promising measure.To be a functional membrane surface molecule, the cdc42gene was screened from the leukocyte layer suppression subtractive cDNA library of the sheep which challenged with Brucella virulent strain and Brucella Strain2vaccine. Cdc42was a important factor in the cell cycle regulation, cell polarity and tumor expression, asa more involved member in Rho family.Cloning of The cdc42full-length cDNA gene sequenceA number of differentially expressed genes were screened from the SSH cDNA library of sheep leukocyte layer and some differentially expressed genes were analyzed in the initial. The partial suspected cdc42gene sequence was screened.out by the sequencing and BLAST method, and the size of sequence fragment was176bp. Two pairs of specific primers were applied to amplified the cdc42full-length cDNA gene sequence by3’and5’RACE PCR. The size of cloned sequence fragment was1609bp, the ORF in cdc42full-length cDNA sequence was576bp, encoding191amino, and its molecular weight was21kDa, the GenBank accession numbers of this sequence was KC425615.The prokaryotic expression of cdc42geneOne pair of specific primer was designed to amplified the cdc42gene complete coding region, cloned to pMD18-T vector, then sub-cloned to the prokaryotic expression vector pET-30a. The recombinant expression vector pET30a-Cdc42was constructed and induced expression by IPTG, The molecular weight of fusion protein was21kDa. The recombinant protein expressed as inclusion bodies.Preparation of the anti-Cdc42monoclonal antibodiesThe fusion protein was purified and BALB/c mices were immunized, the spleen cells of immunized mice were fused with myeloma cells to obtain a hybrid myeloma cells, the annti-Cdc42monoclonal antibody were finally obtained by ELISA detection and then were cloned. The monoclonal antibody is an IgG class Verified by Dot-blot and Western-blot analysis, the monoclonal antibodies have a specifically binding with recombinant proteins and natural protein.