猪链球菌3种毒力因子多细胞表位靶向DC重组蛋白DC3pep-SDZ的构建及安全性检测

Construction and safety testing of Streptococcus suis recombinant DC3pep-SDZ protein containing multi-epitope recognized by dendritic cells

为了研制2、3、9型猪链球菌(SS)通用的重组亚单位疫苗,本研究通过生物信息学软件对SS分泌型核酸酶A(SsnA)、二氢硫辛酰胺脱氢酶(DLDH)、锌转运蛋白(ZnuA)3种毒力因子的蛋白质一级结构、B细胞及辅助T(Th)细胞抗原表位、亲疏水性及抗原性、二级结构进行综合分析后,预测获得了SsnA、DLDH和ZnuA具有亲水性和较好抗原性的B/Th细胞表位,将它们与12聚体树突状细胞靶向肽(DC3pep)序列串联后获得了重组氨基酸序列DC3pep-SDZ。经蛋白质理化分析DC3pep-SDZ分子质量约为44 ku,理论等电点(pI)为4.65,平均亲水性为-0.462,为亲水性蛋白。由生物公司构建重组原核表达载体pET28a-DC3pepSDZ,转化感受态细胞后经IPTG诱导表达后经western blot鉴定重组蛋白以可溶形式表达。表达产物利用亲和层析镍柱纯化,SDS-PAGE结果显示得到44 ku的纯化目的蛋白,纯化浓缩后经BCA蛋白浓度测定试剂盒检测,浓度为5.60 mg/mL。利用不同浓度纯化重组蛋白对2%猪红细胞悬液进行体外溶血试验,并对PK15、IPEC-J2细胞进行毒性试验,检测重组蛋白的生物安全性。体外溶血试验结果显示在5μg/mL~500μg/mL浓度范围内,DC3pep-SDZ蛋白对2%猪红细胞悬液溶血率低于5%;细胞毒性试验结果显示经500μg/mL DC3pep-SDZ蛋白孵育后,PK15与IPEC-J2细胞的相对增殖率(RGR)分别为98.5%和99.6%,与对照组RGR相比均无显著差异。表明,重组蛋白无明显毒性作用。本实验首次构建并表达了重组蛋白DC3pep-SDZ,该蛋白生物安全性良好,为后续分析其免疫保护效果、研发2、3、9型SS重组亚单位疫苗奠定基础。

In order to develop a universal recombinant subunit vaccine aginst Streptococcus suis(SS)type 2,3 and 9,the bioinformatics software was used to analyze the primary structure,B cell and helper T(Th)cell epitopes,hydrophobicity,antigenicity and secondary structure for SS secretory nuclease A(SsnA),dihydrolipoamide dehydrogenase(DLDH)and zinc transporter(ZnuA).The B/Th cell epitopes of SSNA,DLDH and ZnuA with hydrophilicity and good antigenicity were predicted.Connecting epitopes in series with the 12 mer dendritic cell targeting peptide(DC3pep)sequence,the recombinant amino acid sequence DC3pep-SDZ was obtained.Through protein physical and chemical analysis,the molecular weight of DC3pep-SDZ was about44ku,the theoretical isoelectric point(PI)was 4.65,and the average hydrophilicity was-0.462,DC3pep-SDZ was a kind of hydrophilic protein.Constructing recombinant prokaryotic expression vector p ET28a-DC3pep-SDZ,protein expression was induced by IPTG after transforming the competent cells.The expressed product was purified by affinity chromatography on nickel column.The SDS-PAGE results showed that the target protein of 44ku was obtained.After purification and concentration,the concentration was determined as 5.60mg/mL with a BCA protein concentration determination kit.The biological safety of recombinant protein was tested by the different concentration of recombinant proteins were used to perform hemolysis test in vitro in 2%porcine erythrocyte suspension and cytotoxicity test in PK15 and IPEC-J2 cells.The results of hemolysis test in vitro showed that within the range of5μg/mL-500μg/mL of DC3pep-SDZ protein,the hemolysis rate of DC3pep-SDZ protein to 2%porcine erythrocyte suspension was less than 5%.The results of cytotoxicity test showed that at the concentration of 500μg/mL of DC3pep-SDZ protein,the relative growth rates of PK15 and IPEC-J2 cells were 98.5%and 99.6%respectively,indicating that the recombinant protein had no obvious toxic effect.In this experiment,the recombinant protein DC3pep-SDZ was constructed and expressed for the first time.The protein has good biosafety.Our study lay a foundation for the subsequent analysis of its immune protection effect and the development of type 2,3 and 9 SS recombinant subunit vaccine.