罗非鱼无乳链球菌LrrG-Sip融合蛋白免疫原性研究

为探究无乳链球菌LrrG(Leucine-rich repeat protein from GBS)和表面免疫原性蛋白Sip(surface immunogenic protein)串联表达的LrrG-Sip重组融合蛋白的免疫原性,该研究将原核表达的LrrG-Sip重组融合蛋白分别以0.5μg·g~(-1)(R1组)、1.0μg·g~(-1)(R2组)和1.5μg·g-1(R3组)每尾200μL腹腔注射免疫尼罗罗非鱼(Oreochromis niloticus),同时以Sip蛋白(S组)、LrrG蛋白(L组)以及PBS(P组)作为对照。2周后对所有免疫鱼体进行无乳链球菌(Streptococcus agalactiae)人工攻毒,攻毒剂量为其半致死浓度(LD_(50):4.0×108CFU·mL~(-1))。结果显示LrrG-Sip重组融合蛋白R1组对尼罗罗非鱼的相对免疫保护率最高,达89.14%;且免疫后第14和第28天,该组鱼体血清抗体OD_(450nm)值分别达0.63和0.64,均显著高于单一蛋白对照组(S和L组)和PBS组(P0.05);R1组鱼体血清过氧化物酶(POD)和碱性磷酸酶(AKP)活性在上述2个时间点也显著高于其他组(P0.05);但溶菌酶(LZM)和总超氧化物歧化酶(T-SOD)活性与其他组之间差异不显著(P0.05)。初步表明LrrG-Sip重组融合蛋白具有良好的免疫原性,其免疫原性明显优于单个蛋白,且能有效减少免疫剂量。

Immunogenicity analysis of LrrG-Sip fusion protein of Streptococcus agalactiae in tilapia

To evaluate the immunogenicity of LrrG-Sip recombinant fusion protein,we immunized Nile tilapia (Oreochromis niloticus) by intraperitoneal (IP) injection with 200 μL LrrG-Sip recombinant fusion protein at dose of 0.5 μg·g-1 (R1 group),1.0 μg·g-1 (R2 group) and 1.5 μg·g-1 (R3 group),respectively. Meanwhile,control groups of Nile tilapia were IP injected with 200 μL Sip protein (1.0 μg·g-1,S group),200 μL LrrG protein (1.0 μg·g-1,L group) and 200 μL sterile phosphate buffered saline (PBS,P group),respectively. On 15th day,all of the immunized fish (groups of R,S,L and P) were challenged with Streptococcus agalactiae by IP injection at LD50 dose of 100 μL (containing 4.0×108 CFU·mL-1). Results show that the relative percent survival (RPS) of fish from group R1 (0.5 μg·g-1) was the highest (89.14 %). The OD450 nm values of serum antibody of fish from group R1 were 0.63 and 0.64 on 14th day and 28th day post-immunization,respectively,significantly higher than those of the other groups (S and L)(P<0.05).The activity of peroxidase and alkaline phosphatase of fish serum from this group was also significantly higher than that of the other groups at the above two time points (P<0.05). There was no significant difference in the activity of lysozyme and total superoxide dismutase of fish serum among different protein immunization groups(P>0.05). The results suggest that LrrG-Sip fusion protein has better immunogenicity than single protein LrrG or Sip,which can also reduce the injection dose effectively.