罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析

表面免疫原性蛋白(Surface Immunogenic Protein,Sip)是B群链球菌(Group B Streptococcus,GBS)的一种表面蛋白,在GBS多种血清型的菌株中均有表达。研究从实验室分离到的罗非鱼无乳链球菌广东株的基因组DNA中扩增出Sip基因,构建原核表达载体pColdII-Sip,转化大肠杆菌(Escherichia coli)BL21(DE3)菌株。经诱导表达、SDS-PAGE电泳检测显示重组蛋白主要以可溶形式表达。重组蛋白用亲和层析的方法纯化,蛋白纯度达98%。纯化后的重组蛋白免疫罗非鱼(Oreochromis niloticus,GIFT strain)以分析其免疫原性。受免鱼免疫14d后进行人工攻毒试验,免疫组的相对保护率为70%—87%。酶联免疫吸附试验(Elisa)显示受免鱼对重组蛋白产生了较好的免疫应答,免疫剂量为3μg/g和5μg/g时受免鱼血清抗体滴度可达1:128000。研究结果显示重组蛋白Sip具有较强的免疫原性和保护作用,Sip基因可作为罗非鱼链球菌基因工程亚单位疫苗候选基因。

CLONING, EXPRESSION AND IMMUNOGENICITY ANALYSIS OF SURFACE IMMUNOGENIC PROTEIN (SIP) OF TILAPIA STREPTOCOCCUS AGALACTIAE

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,KpnI and HindⅢ.The amplified product,as well as pColdII,were digested respectively.After purification and ligation,the ligated DNA was transferred into E.coli BL21(DE3) strain.The recombinant expression vector pColdII-Sip was selected and then induced to express by 1 mmol/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 recombinant 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 3 μg/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.