B族链球菌表面蛋白LrrG的分段表达及其免疫原性和功能研究

Expression and functional study of fragment pepfides of group B streptococcal LrrG protein

目的研究LrrG蛋白不同片段的免疫原性及保护功能，为B族链球菌（GBS）蛋白疫苗及组分疫苗的研究奠定基础。方法采用生物信息学的方法预测LrrG蛋白的抗原表位，结合该蛋白的一级结构特征构建3个多肽片段（Gn、Gr、Gc）并重组表达；通过免疫动物及萃取组织液检测其免疫原性；体外调理吞噬试验检测各多肽片段的保护功能。结果成功构建和表达纯化了Gn、Gr、Gc多肽片段，且3个多肽片段均可在小鼠血清诱导产生较高水平的特异性IgG抗体，Gn免疫原性好于Gr、Gc。Gr、Gc30腭剂量的免疫原性均好于其10μg和50μg剂量。除了Gr在肺、直肠组织产生的IgA抗体有较明显升高外，Gn、Gc对黏膜及血清IgA无明显刺激作用。体外调理吞噬试验表明3个多肽片段的抗体均具有促进吞噬细胞对GBS的吞噬功能。Gr、Gc的调理吞噬活性高于Gn，与B细胞表位预测结果相符。结论重组多肽片段均具有较强的免疫原性，在一定免疫剂量范围内，血清特异性IgG水平呈剂量依赖关系；皮下免疫对黏膜免疫影响不大；调理吞噬作用可能是LrrG蛋白具有保护作用的机制，3个多肽片段均具有潜在保护作用，支持将其作为GBS蛋白疫苗及组分疫苗的候选成分。

Objective To construct fragment peptides of group B streptococcal LrrG protein and study their immunogenicity and protective function. Methods Three fragments（Gn, Gr, Gc） of LrrG protein were con- structed according to the prediction result of LrrG protein＇ s antigenic sites and the characteristics of its primary structure. The recombinant fragments were expressed and purified. CD1 mice were immunized with Gn, Gr, Gc separately to test their immunogenicity. Perfusion-extraction method was used to obtain lung, rectal and vagina specimens for antibody detection. The IgG and IgA levels in mice serum and issues were tested by ELISA. Op-sonophagocytosis test in vitro was used to test the protective function of anti-Gn, Gr, Gc serum. Results Gn, Gr, Gc were successfully expressed and purified with affinity chromatography. ELISA data showed that Gn, Gr and Gc could elicit significantly higher level of IgG in serum of immunized mice than that of control group （P〈0. 001）. 30 μg dose of Gr and Gc can elicit higher level of specific IgG than that of 10 μg and 50 μg doses. Except that Gr elicited higher IgA level in lung and rectum, Gn and Gc have little influence on IgA level of serum and tissues. Opsonophagocytosis tests showed that anti-serum of Gn, Gr and Gc had opsonophagocytic activity than control serum and negative control, and their infection index of phagocytes were 617.76,907.83, 906.30, 225.08, 247.04 respectively after incubating with phagocytes and GBS for 30 min, which were also confirmed by counting the number of GBS on blood agar after lysis of phagocytes. After 3 h incubation the infection index decreased, which showed the killing function of phagocytes. Gr and Ge have better opsonophagocytic activity than Gn, though Gn has higher IgG level, but the result is accorded with the prediction. Conclusion Gn, Gr and Ge are immunogenie. The specific IgG level is dependent on the dose of immunized protein to some extent, and beyond the extent it may inhibit immune response. Immunization from subcutaneous route seems to have little influence on mueosal immunity, which can provide experimental data for further mueosal immunity. The opsonophagocytie activity may be the proteetive mechanism of LrrG protein. Antiserum to Gn, Gr and Ge have potential proteetive function, which supports them to be components of GBS vaccine.