An Indirect ELISA Method Established For Detecting Streptococcus Agalactiae Mediated By High Affinity Phages With Sip Protein

Cattle mastitis is one of the world’s most common and most damaging diseases in cattle industry. It not only influences the amount of milk production and its quality, but also leads to the elimination of sick cows and causes enormous economical losses. Moreover, pathogenic microorganisms in the contaminated milk can bring harms to human’s health. Streptococcus agalactiae is one of the main pathogenic microorganisms of cow mastitis. Its surface immune proteins(Sip) expressed in all nine kinds of serotypes of bacteria. As one of bacterial adhesion and colonization factors, it has better immune protection reaction and antigenicity and can be used as an important candidate of vaccine antigens. Comparison of the predicted amino acid sequences of Sip protein from nine serologically distinct strains clearly indicated that this protein was highly conserved. As a result, it is feasible to develop a rapid and effective method to detect Streptococcus agalactiae infection for improving the quality and safety of dairy product, as well as benefit to human’s health.This study successfully amplified Streptococcus agalactiae Sip gene and cloned it into prokaryotic expression vector p GEX-6P-1, resulting in a recombinant plasmid p GEX-6p-1-Sip, followed by expression in Rossetta with the optimal conditions of final concentration of 1.0 mmol/L IPTG, and 37℃ for 5 h. Its molecular mass was 59.8 Ku. The recombinant protein was purified and used as target protein for phage screening.Subsequently, Random 12-mer Phage Display Peptide Library was used to pan the Sip protein for four rounds. GBS Sip was packaged with the quantity for 10 μg in the first round, then the next round in half. The phage was packaged with the quantity for 1.5 x 1011 CFU/μL per round. Finally, ten monoclonal phages were selected randomly and characterized high affinity with Sip protein.Then an indirect ELISA method for detecting Streptococcus agalactiae mediated by high affinity phages with Sip protein was established, with positive phage as the first antibody, M13 Pc Ab as the second antibody. Optimization results as follows: package concentration of antigen was 10 CFU/m L/hole, concentration of phage was 108 PFU/m L and incubating time was 60 min, coating way of antigen was 4℃ over night after 37℃ for 2 h, sealing agent was the 1.0% BSA and incubating time was 3 h, concentration of M13 Pc Ab was 1‰ and incubating time was 45 min, enzyme-conjugated antibody dilution degrees was 1:4000 and incubated for 45 min. The sensitivity of this method to detect was 10 CFU/m L and only reacted with positive reference samples. Fifty four milk samples of cow mastitis were tested and the positive rate was up to 14.286%. This experiment combined phage display techniques with indirect ELISA detection method to provide a new solution to detect Streptococcus agalactiae simply and effectively.