罗非鱼源无乳链球菌溶血素基因体外表达及其免疫原性

摘要

Streptococcus is a genus of spherical gram-positive bacteria that can infect a wide range of hosts. Streptococcal diseases of cultured fish caused by Streptococcus agalactiae have been frequently reported in South China, which incurred catastrophic economic losses. Vaccine is one of effective ways to control and eradicate infectious diseases, and so far no effective subunit vaccine for S. agalactiae diseases has been developed. To test whether Hemolysin (Hly) is a potential candidate for vaccine development of Oreochromis niloticus, we cloned hly gene based on bioinformatics analysis and constructed and expressed it. The open reading frame of hly gene contains 1335 bp that encodes 444 amino acid residues. Alignment analysis indicated that the Hly protein was highly homologous to Hly proteins from other Streptococcus(99%). SDS-PAGE confirmed the expression of Hly (51.7 kD) in Escherichia coli BL21(DE3). Then, the recombinant Hly protein was purified by affinity chromatography, which was used for vaccination in Tilapia, O. niloticus. Immunogenicity was confirmed by subsequent western blotting. Enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that Hly produced an observable antibody response in all vaccinated fish, and the maximum antibody titers in the sera reached 1:4096. The RPS value for the Sip vaccine was 70. Tilapia vaccinated with Hly was highly resistant to the infection of the contaminant S. agalactiae. These results indicate that Hly is an effective vaccine candidate against S. agalactiae for tilapia, O. niloticus.%为研究罗非鱼源无乳链球菌溶血素(Hemolysin, Hly)对鱼体的免疫保护作用，根据已获得的无乳链球菌ZQ0910全基因组序列设计引物扩增hly基因，定向克隆于原核表达载体pET-28a中，构建原核重组质粒pET-28a-hly，经IPTG诱导表达后，制成亚单位疫苗免疫吉富罗非鱼，并分析疫苗的免疫保护力。结果显示，hly基因产物大小1335 bp，编码444个氨基酸，经测序与GenBank报道的链球菌属Hly氨基酸序列同源性可达99%。经IPTG诱导表达后，SDS-PAGE分析可见一条51.7 kD的特异条带；Western blotting分析结果说明表达的Hly蛋白能与His-Tag单抗特异性结合；制备的亚单位疫苗免疫鱼体后第14天即可检测到抗体产生，并在第28天达到峰值，抗体效价为1∶4096，免疫保护率为70%。由此证实，该亚单位疫苗有望成为预防由无乳链球菌引起的罗非鱼链球菌病的基因工程类疫苗。