Prokaryotic Expression And Character Analysis Of Streptococcus Iniae CAMP Factor

In this study, total bacterial DNA of was extracted from Streptococcus iniae (S. iniae) of Guangxi isolates DGX07. CAMP factor gene (cfi) was amplified and introduced into pMD19-T. Cfi was sequenced and bio-softwares were employed for sequence analysis. Cfi with two restriction sites was cloned into pMD19-T and then introduced into pET32a(+). Positive pET32a(+) was introduced into bio-engineered Escherichia coli BL21(DE3) and conditions for over-expression were optimized. New Zealand white rabbits were immunized with recombinant CAMP factor and CAMP factor anti-serum was purified and used in S. iniae CAMP factor detecting. CAMP factor anti-serum were employed to perform the immunoglobulin binding. Epithelioma papulosum cyprini (EPC) cell adherence and invasion, and co-hemolysis with recombinant Staphylococcus aureus sphingomyelinase on red blood cells of several species were also performed.Sequence analysis of Streptococcus iniae cfi showed that CAMP factor belonged to CAMP factor superfamily and was homogenous to its counterparts in other streptococcus(Streptococcus canis 63% highest), but with a relatively low similarity to them. CAMP factor consisted 60.1% variable amino acids, suggesting that it could not be a conservative protein. CAMP factor had a signal peptide sequence and one potential N-glycosylation site and sixteen phosphorylation sites. Recombinant CAMP factor on SDS-PAGE was was about 48kDa. The optimization for over-expression was under 37℃ with 0.8mmol/L for 5h. The titer of this anti-serum was 1:32. Antigenicity of CAMP factor against this serum was verified by Western-blot. Western-blot of CAMP factor anti-serum against ultrasonic treated S. iniae indicated that S. iniae under natural growing environment would secrete certain amount of CAMP factor. In the vitro cell assay, CAMP factor-neutralized-S.. iniae showed an decreased adherence and invasion capability against EPC cells (58.4% and 42.7% respectively), indicating that CAMP factor played a remarkable role in facilitating S. iniae on the internalization into epithelium. Western-blots showed that CAMP factor could nonspecifically bind to the IgG in a dose-dependent manner, resulting in the decreased capacity in antibody-antigen reaction. Results of co-hemolysis indicated that CAMP factor showed time-dependent and dose-dependent manners on the co-hen)olysis to sheep, pig and rabbit red blood cells, whereas fish red blood cells were suggested otherwise. This results indicated that CAMP factor in S. iniae might lose hemolytic ability to fish red blood cells, but compensatorily functioned in the IgG Fc region binding as well as epithelium adherence and invasion.