水生生物学报2012，Vol.36Issue(4)：626-633,8.DOI:10.3724/SP.J.1035.2012.00626
罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析
CLONING, EXPRESSION AND IMMUNOGENICITY ANALYSIS OF SURFACE IMMUNOGENIC PROTEIN (SIP) OF TILAPIA STREPTOCOCCUS AGALACTIAE
摘要
Abstract
Surface immunogenic protein (Sip) is a kind of immune related protein that exists in group B Streptococcus, which expresses in many serotypes of GBS. In this present study, Sip gene was amplified from the genomic DNA of a Streptococcus agalactiae strain isolated from sick pond-cultured tilapia in Guangdong province, China. The Sip gene contained a 1305 bp Open Reading Frame (ORF), which encoded 434 amino acids. The molecular mass of the deduced amino acid sequences was 56 kD. Blast analysis showed that it shared high identities (100%) with Sip sequences of human GBS registered in GenBank, while lower identities (< 50%) was found compared with other species of Streptococcus. Prediction of epitopes by DNAStar software showed that the deduced amino acid sequence of this Sip gene could form 29 epitopes indicating strong immunogenicity of the Sip. Prokaryotic expression vector pColdII was used to construct a recombinant expression vector pColdII-Sip. Employing PCR method, the cloned Sip gene was modified with primers carried two specific restriction enzyme digested sites, Kpnl and HindIII. The amplified product, as well as pColdll, were digested respectively. After purification and ligation, the ligated DNA was transferred into E. coli BL21 (DE3) strain. The recombinant expression vector pColdll-Sip was selected and then induced to express by 1 rnmol/L IPTG for 24h at 15℃. SDS-PAGE analysis and Western blot showed that a specific band of protein about 57 kD in molecular weight was obtained. SDS-PAGE analysis of the induced samples found that the target protein was detected only in the supernant rather than in precipitate, suggesting that it was solubly expressed. The purification of the protein was up to 98% after purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the recombinant protein, tilapia (Oreochromis niloticus, GIFT strain) was immunized by intraperitoneal injection of the recornbinant protein. Three immune dosages, 1 μg/g, 3 μg/g and 5 μg/g were used. Two weeks after immunity, tilapia was challenged by artificial infection of GBS GDzl strain, which had been previously isolated and confirmed to be tilapia pathogen by our lab. The recorded relative percent survival (RPS) of the vaccinated groups ranged from 70% to 87%. Enzyme-linked immunosorbent assay (ELISA) showed that the recombinant protein could induce strong immune response of the tested fish. The serum titers of the fish immunized with 1 μg/g of Sip protein was 1 : 16000, and the serum titers of the fish immunized with 3μg/g or 5 μg/g of Sip protein reached 1 : 128000. The result showed that the recombinant protein possessed strong immunogenicity, and Sip gene could be a candidate gene for genetic engineering vaccine.关键词
罗非鱼/无乳链球菌/表面免疫原性蛋白/原核表达/免疫原性/抗体/相对保护率Key words
Tilapia/ Streptococcus agalactiae/ Surface immunogenic protein/ Prokaryotic expression/ Immunogenicity/ Antibody/ Relative percent survival分类
生物科学引用本文复制引用
黎炯,叶星,可小丽,卢迈新,迟妍妍,田园园..罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析[J].水生生物学报,2012,36(4):626-633,8.基金项目
国家高技术研究发展计划(863计划)(No.2011AA100404) （863计划）
现代农业产业技术体系专项资金资助(CARS-49) （CARS-49）
广东省海洋渔业科技推广专项(No.A201101C02)资助 （No.A201101C02）
