肺炎链球菌自溶酶蛋白抗原表位的预测、克隆及重组表达

目的用生物信息学的方法预测肺炎链球菌自溶酶蛋白(LytA)可能的抗原表位,合成带有linker(Gly4 Ser1)3序列的融合基因片断(l1l2),并在原核系统表达。方法利用多种生物信息学软件,在二级结构预测抗原表位分值的基础上结合单参数分析结果,预测并设计LytA可能的重组抗原表位(L1L2)。扩增该表位基因l1l2,利用分子克隆技术构建重组质粒。经测序鉴定后,转入宿主菌JM109中原核表达L1L2多肽片断与载体标签蛋白还原性谷胱苷肽(GST)的融合蛋白GST-L1L2。结果预测表明序列2～28(EINVSKLRTDLPQVGVQPYRQVHAHST)、序列98～112(FMTDYRLYIELLRNL)分别为可能的B细胞和T细胞抗原表位。用PCR技术合成了l1l2片段,构建重组质粒pGEX-4T-1-l1l2,成功转化大肠杆菌JM109。结论经测序验证成功构建了重组质粒pGEX-L1L2,并转化大肠杆菌JM109,SDS-PAGE分析显示该重组质粒在原核系统中得到了表达,为后继多肽疫苗研究奠定了基础。

Prediction, recombination and expression of the antigenic spitopes in autolysin LytA of Streptococcus pneumoniae

To predict the antigenic epitopes in autolysin LytA of Streptococcus pneumoniae with bioinformatics method so as to design the useful vaccine for pneumonia, the fusion gene fragment (l1l2) carrying linker Gly4-Ser1 was synthesized and expressed on prokaryotic expression system. Using various bioformational softwares and network servers with parameter and secondary structure analysis to identify the antigenic spitopes in terms of value scores, the methods were based on the comprehensive way. The B cell antigenic epitope prediction tools were used to compare the antigenicities of different serotypes of LytA protein and the genes of the recombinant epitopes were constructed by PCR. The PCR product was then cloned into recombinant vector which was induced to E.coli host strain JM109.to express l1l2 peptide fragment and the fusion protein GST-l1l2. It was demonstrated that sequence 2-28 (EINVSKLRTDLPQVGVQPYRQVHAHST) and sequence 98-112 (FMTDYRLYIELIRNL) might be the possible antigenic epitopes for LytA on B cells and T cells respectively. Different serotypes of LytA protein were showed to have the same spatial antigenic spitopes. The recombinant protein GST-l1l2 was found to be expressed in E.coli host strain JM109 and this was checked by SDS-PAGE electrophoresis. These results can provide basis for the further studies of polypeptide vaccine against pneumococcal infections.