罗非鱼无乳链球菌LrrG蛋白的原核表达及免疫原性分析

Prokaryotic expression and immunogenicity analysis of LrrG protein of Streptococcus agalactiae isolated from tilapia

LrrG蛋白是无乳链球菌较保守的表面蛋白之一。为获得罗非鱼源无乳链球菌LrrG蛋白并探讨其在罗非鱼体内的免疫原性,本实验根据GenBank中已报道的人源无乳链球菌LrrG基因序列,设计特异性引物,扩增获得罗非鱼源无乳链球菌的LrrG基因。分析表明,其ORF为2 361 bp,编码786个氨基酸,与人源无乳链球菌LrrG基因核苷酸序列的相似性高达98.48%。LrrG蛋白含有3个保守的LRR结构域,并可形成多个抗原表位。将LrrG基因片段克隆转入原核表达载体pET-32a(+),构建重组质粒pET-32a(+)/LrrG,E.coli BL21(DE3)22℃诱导表达6 h。SDS-PAGE显示,诱导表达蛋白的分子量为108.9 ku,并且该重组蛋白以可溶和包涵体2种形式存在。经His Bind亲和柱纯化及超滤管浓缩后,LrrG可溶蛋白浓度达3.40 mg/mL。鱼体注射免疫实验表明,LrrG可溶蛋白对罗非鱼的相对免疫保护率达69.28%,且免疫后4周的血清抗体滴度为1∶800。该研究为深入探讨无乳链球菌LrrG蛋白作为罗非鱼基因工程疫苗的潜在应用价值奠定了基础。

Tilapia is one of the important species in aquaculture in the world. In recent years,tilapia aquaculture has been badly damaged by streptococcusis. Streptococcus agalactiae is one of the major causative agents,which has caused severe economic losses in many species of freshwater,marine and estuarine fish worldwide. Vaccine is one of the main development future strategies to prevent fish streptococcusis. Some surface proteins of S. agalactiae have been documented and proved to be the ideal antigen candidates of protein vaccines,which included LrrG protein. The LrrG protein is one of the conserved surface proteins of S. agalactiae,which exists in all kinds of serotype of S. agalactiae strains. The LrrG protein of S. agalactiae isolated from human has been verified to protect mice from S. agalactiae infection. However,it has not been reported whether the LrrG protein from fish S. agalactiae isolates possessed the similar effect. In order to obtain plenty of LrrG protein and explore its immune protection in tilapia,in this study we designed the specific primers and cloned the LrrG gene sequence from tilapia virulent S. agalactiae strain according to the reported LrrG gene in GenBank of human S. agalactiae. The sequencing results showed that the ORF of the cloned LrrG was 2 361 bp in length and encoded 786 amino acids and contained 3 conserved LRR（ Leucine-rich repeat） domains. The colis accounted for 68. 83% in its secondary structure. NCBI Blast indicated that the homology of LrrG gene sequences of S. agalactiae from tilapia and human was 98. 48%. The LrrG protein could form antigenic epitope based on the analysis of three indexes,i. e. hydrophilicity,surface probability and antigenic index. This suggests that the LrrG protein may have potential immunogenicity. The LrrG fragment was subcloned into pET-32a（ ＋） vector and expressed in E. coli BL21（ DE3） by IPTG induction at 22 ℃. SDS-PAGE showed the molecular weight of expressed protein was 108. 9 ku,which is equal to the expected protein size. The recombinant protein existed as soluble protein and inclusion bodies. The soluble protein was highly purified by Ni-chelating affinity chromatography and the concentration could reach 3. 40 mg / mL by ultrafiltration. Preliminary experiment showed that the relative percent survival of tilapia immunized with LrrG protein against tilapia streptococcicosis was 69. 28%. ELISA analysis indicated the serum antibody titer of post-immuned tilapia for 4 weeks was 1 ∶ 800. These findings provide the useful information for further investigation to evaluate the potential value of the LrrG protein as tilapia genetic engineering vaccine.