Cloning,Prokaryotic Expression Of Brucella Abortus SP41Gene And Preparation Of Antiserum

This research is to clone the SP41gene of Brucella abortus. Construct prokaryoticexpression vector of pET-32a(+)-SP41gene and prepare antiserum using the fusion protein asantigen for the ELISA detection of Brucellosis.One pair of specific primers to SP41gene was designed and synthesized based on thepublished sequence in GenBank. SP41gene was cloned from genomic DNA of Brucella A19vaccine by PCR. PCR product was recovered and cloned into pEASY-T3vector. PCRamplification and endonuclease digestion results confirmed that the SP41gene had beeninserted into the cloning vector pEASY-T3. DNA Sequencing analysis showed that theamplified fragment was composed of1302nucleotides and encodes433amino acids.Compared with the reported SP41gene from whole genome DNA sequence of2308,A-13334, S19,9-941, the amino acid identity was100%, and the amino acid identity was99.8%comparing with ATCC, CCM4915, M28, M5-90, NI.The gene was cloned into the prokaryotic expression vector pET-32a(+). After restrictionendonuclease analysis and sequencing following PCR, it was confirmed that the expressionvector pET-32a-SP41was constructed successfully. The pET-32a(+)-SP41was transformedinto Escherichia coli BL21cells (processed by DE3). Discovered through the exploration ofthe induction conditions, IPTG concentration has little effect on protein expression. Theexpression effect was the best under37℃, induced6h. The expressed product was identifiedby Western blot and suggested that the recombinant protein has good antigenicity. Thepurified recombinant protein vaccinated to healthy male rabbits, after4rounds of vaccination,the anti-serum antibody levels reached to1:51200with the method of iELISA.We have the purified recombinant SP41coated on the ELISA plate for detecting theclinical brucellosis bovine serum, which provided a solid foundation for the exploring ofSP41as a diagnostic protein.