论文部分内容阅读
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。
OBJECTIVE: To design a recombinant polypeptide vaccine against four serotypes of Streptococcus group A, Streptococcus group A, Streptococcus pneumoniae M1,3,6,18 which cause rheumatic fever and to synthesize and clone the nucleotide sequence of the recombinant polypeptide vaccine. Methods Four serotypes M serotypes of Streptococcus group A 1,3,6,18 were obtained from the CDC database. According to the reported and combined bioinformatics methods, each serum was screened Type of specific sequence with human histone no homology of the segment, according to the 1-3-6-18 type in series, at the C-terminus of the tandem sequence plus a total of four serotypes Of human tissue protein cross-epitope, and has a certain immunogenicity of the conserved sequence J14. BlastP alignment, hydrophilicity analysis, secondary structure analysis, spatial structure analysis, and B cell epitope prediction of human histone proteins were carried out on the amino acid sequence of the designed peptide vaccine. A set of oligonucleotide primers was designed by using DNAWORK2.0 online software. OverlapPCR method was used to synthesize the nucleotide sequence of the polypeptide vaccine. BamHⅠ and HindⅢ restriction sites were introduced into the 5 ’and 3’ end respectively. After double digestion, cloned into pUC18 vector and sequenced. Results The software analysis showed that the designed peptide vaccine has no homology with human tissue protein and has good hydrophilicity. The secondary structure and spatial structure are similar to the native M protein, and may exist in every serotype segment B cell epitope. The DNA sequence of the polypeptide vaccine was successfully amplified and a recombinant cloning vector was obtained which was completely consistent with the designed sequence. Conclusion A tetravalent recombinant group A streptococcal peptide vaccine has been successfully designed and laid the foundation for its recombinant expression and immunogenicity.