利用噬菌体展示技术构建Mhp168株Hsp70随机肽库

Construction of gene-targeted phage random peptide library for Mycoplasma hyopneumoniae 168 strain Hsp70 protein

[目的]构建Mhp168株Hsp70 C端基因的噬菌体展示随机肽库。[方法]扩增猪肺炎支原体168株热休克蛋白Hsp70 C端1803 bp的DNA序列。回收Hsp70 DNaseⅠ消化产物中80~150 bp的DNA片段与pC89载体连接。辅助噬菌体超感染转化重组噬粒的E.coli XL1-Blue细胞。检测肽库的多样性,测定库容。[结果]在噬菌体表面展示出Hsp70 C端基因的随机片段融合蛋白。随机肽库库容约为9.9×10^3,滴度约为1.5×10^13 PFU/ml。[结论]所构建的随机肽库可以满足Hsp70构象型抗原表位筛选。

[Objective]Construction of gene-targeted phage random peptide library for Mycoplasma hyopneumoniae(Mhp)168 strain Hsp70 C-terminal.[Method]The target sequence of Mhp 168 strain Hsp70 C-terminal was amplified by PCR.The result of sequencing indicated that the Hsp70 C-terminal gene of Mhp 168 strain was 1803 bp in length.The PCR products of Hsp70 were digested by DnaseⅠ.The fragments between 80 and 150 bp were reclaimed by agarose gel.The purified and recoveried random fragments were ligated with the pc89 vectors.The recombinants were transformed into competent E.coli XL1-Blue cell,and then rescued with help phage VCSM13.The size and diversity of the library were checked.[Result]The gene-targeted random peptide library of Hsp70 was constructed.The capacity of the library was 9.9×10^3 pfu,and the titers was 1.5×10^13 PFU/ml.[Conclusion]These results showed that the library may meet the demands of mapping and screening the Hsp70 epitopes.