变异链球菌PAc、Glu重组蛋白的抗体制备及体外抑菌活性研究

Preparation and in vitro bacteriostatic activity of antibodies against recombinant protein of PAc and Glu of Streptococcus mutans

目的:制备变异链球菌细胞表面蛋白抗原PAc及葡糖基转移酶GTF-B的羧基端葡聚糖结合区Glu蛋白的抗体,并研究其体外对变异链球菌的影响。方法:含有PAc及Glu基因片段的质粒转化E.coli BL21(DE3)得到宿主菌,在18℃、终浓度为1 mmol/L的IPTG条件下诱导过夜,并通过His-Tag标签纯化目的蛋白。纯化的目的蛋白联合弗氏佐剂免疫新西兰大白兔,ELISA检测血清中抗PAc及抗Glu IgG水平,并研究抗体对变异链球菌的影响。结果:成功表达纯化PAc和Glu蛋白。PAc蛋白二免后抗血清效价为1:512000,Glu蛋白二免后抗血清效价为1:32000。两种蛋白免疫后在血清中产生的特异性抗体对变异链球菌的生长有一定的抑制作用。结论:PAc、Glu蛋白免疫产生的特异性抗体对变异链球菌生长具有抑制作用,有进一步开发为抗变异链球菌生物制剂的价值。

Objective:To prepare polyclonal antibody against cell surface protein antigen(PAc)and Glu of glucosyltransferase B(GTF-B)of Streptococcus mutans(S.mutans),and to study its effect on S.mutans in vitro.Methods:The plasmids encoding PAc and Glu genes were transformed into E.coli BL21(DE3).The target protein expression was induced at 18℃ overnight by IPTG with a final concentration of 1 mmol/L,and was purified by His-Tag.New Zealand white rabbits were immunized with the purified target protein along with Freund’s adjuvant.The levels of anti-PAc and anti-Glu IgG in serum were detected by ELISA,and the effect of antibodies on S.mutans were studied.Results:The PAc and Glu proteins were successfully expressed andpurified.The antiserum titers of PAc and Glu were 1:512000 and 1:32000 respectively after the second immunization of the rabbits and the specific antibodies in serum inhibited the growth and reproduction of S.mutans.Conclusion:The specific antibodies produced in the rabbit serum following the PAc and Glu immunization can inhibit the growth of S.mutans,which has the value of further developing anti-S.mutans biological agents.