中文摘要：

目的构建免疫球蛋白结合分子金黄色葡萄球菌蛋白A（SPA）和链球菌蛋白G（SPG）单结构域随机组合噬菌体展示文库,为研究其分子功能与结构的关系奠定基础。方法 PCR扩增制备SPA的A、B、C、D、E结构域和SPG的B2结构域核苷酸片段,在片段两端通过引物引入XbaⅠ酶切位点和保护碱基,3＇端引入3个氨基酸大小的随机连接肽核苷酸序列,各结构域核苷酸片段经XbaⅠ酶消化,在T4连接酶作用下随机相互连接并克隆于噬菌体展示载体pCANT-AB5S,克隆产物转化E.coliDH5α,提取原代库质粒转化E.coliTG1,经M13K07辅助噬菌体拯救,构建成SPA和SPG单结构域随机组合噬菌体展示文库。结果该噬菌体展示文库库容量为1.87×107cfu,滴度为1.3×1014TU/ml,文库的片段插入率近100%,其中插入多个结构域的阳性克隆占21.7%,序列分析显示各结构域之间的连接和随机连接肽序列具有随机性。结论成功地构建了SPA和SPG单结构域随机组合噬菌体展示文库,该文库在库容量、多样性和随机性方面满足体外分子进化研究的要求。

英文摘要：

Objective To construct a phage-displayed random combinatorial library with single domains of staphylococcal protein A（SPA） and streptococcal protein G（SPG） ,laying the foundation for studying the relationship between the function and structure of these Ig-binding molecules.Methods That the gene fragments encoding the A,B,C,D and E domains of SPA and the B2 domain of SPG were generated by PCR amplification using the primers which introduced recognition site for XbaⅠin both ends and a random linking sequence in 3＇ terminal,which encoded random linking peptide containing 3 amino acids.All fragments of these domains were digested by XbaⅠenzyme and ligated randomly with each other before recombined into the phagemid vector pCANTAB5S.The recombinants were transformed into E.coli DH5α,extracted from E.coli DH5α,and re-transformed into E.coli TG1,and rescued by the helper phage M13K07,then a phage-displayed random combinatorial library of SPA and SPG single domains was established.Results The combinatorial library consisted of about 1.87 × 107 clones and the titer of the phage library was 1.3 × 1014 TU/ml ,nearly 100% clones in the library were positive for insertion and about 21.7% positive clones contained more than two inserted domains.Sequence analysis showed that single domains ligated randomly and the nucleotide acids encoding random linking peptide distributed randomly.Conclusion The phage-displayed random combinatorial library of SPA and SPG single domains has been constructed successfully.The capacity,variety and randomicity of the library meet the requirements of in vitro molecular evolution study.