【摘要】 目的设计针对可引起风湿热的A群链球菌M1、3、6、18四个血清型的重组多肽疫苗,合成并克隆该重组多肽疫苗的核苷酸序列。方法从美国疾病预防和控制中心(CDC)数据库获得A群链球菌1、3、6、18四个血清型M蛋白型特异性序列,根据文献报道并结合生物信息学的方法,筛选每一个血清型的特异性序列中与人体组织蛋白无同源性的区段,将其按1-3-6-18型的顺序串连,在串联序列的C-端加上一个四个血清型所共有的与人体组织蛋白无交叉表位、且具有一定免疫原性的保守序列J14。对设计的多肽疫苗的氨基酸序列进行与人体组织蛋白的BlastP比对、亲水性分析、二级结构分析、空间结构分析以及B细胞表位预测。采用DNAWORK2.0在线软件设计一组寡核苷酸引物,OverlapPCR方法合成该多肽疫苗的核苷酸序列,并在5′端和3′端分别引入BamHⅠ和HindⅢ的酶切位点,将该序列双酶切后克隆至pUC18载体上,并测序。结果软件分析显示,设计的多肽疫苗与人体组织蛋白无同源性,有较好的亲水性,二级结构与空间结构均与天然M蛋白相似,在每一个血清型的区段都可能存在B细胞表位。成功扩增出了该多肽疫苗的DNA序列,并获得了一个与设计序列完全一致的重组克隆载体。结论已成功设计了一种四价重组A群链球菌多肽疫苗,为其重组表达和免疫原性的研究奠定了基础。更多还原

【Abstract】 Objective To design a recombinant polypeptide vaccine specific to group A streptococcus serotypes M1, 3, 6 and 18, which may cause rheumatic fever, and synthesize and clone the gene sequence of the designed vaccine. Methods The serotypespecific amino acid sequences of M protein of group A streptococcus were obtained from the CDC emm typing center website, from which the fragments without homologies to human tissue proteins were screened by the methods reported in documents as well as bioinformatics method. A recombinant polypeptide vaccine was designed by linking the screened fragments and a common conservative sequence J14 which showed a certain immunogenicity but no cross reaction with human tissue protein in the order of M1-3-6-18J14, and analyzed for homology of amino acids to that of human tissue protein by BlastP, then analyzed for hydrophility, secondary structure and spatial structure and predicted for B cell epitopes. A group of oligonucleotides were designed by DNAWORK2. 0 software, based on which the oligonucleotide sequence of designed vaccine was synthesized by overlap PCR, in which the restriction sites of BamHⅠand Hind Ⅲ were introduced at 5′ and 3′terminus respectively. The synthetic sequence was digested with BamHⅠand Hind Ⅲ and cloned into cloning vector pUC18, and the constructed recombinant plasmid was identified by sequencing. Results The designed vaccine showed no homology to human tissue protein. However, the vaccine showed good hydropility, and its secondary and spatial structures were similar to those of natural M protein. B cell epitopes might exist in the fragments of each serotype. The DNA sequence of designed vaccine was successfully cloned, and a recombinant cloning vector carrying designed DNA sequence was obtained. Conclusion A tetravalent recombinant group A streptococcal polypeptide vaccine was successfully designed, which laid a foundation of study on expression and study on immunogenicity of the vaccine.更多还原