猪肺炎支原体P97基因随机肽库的构建

Construction of Phage Gene-targeted Random Peptide Library of P97 Gene of Mycoplasma hyopneumoniae

目的:构建能用于抗原表位筛选的猪肺炎支原体(Mhp)P97基因C-端序列噬菌体随机肽库。方法:以MhpZ株(强毒)基因组DNA为模板,通过PCR扩增获得MhpP97基因的C-端部分序列,扩增产物用DNaseⅠ消化并回收50bp^100bp的随机片段,将回收的随机片段插入pC89pⅧ型噬菌粒载体中,转化大肠杆菌XL1-Blue,辅助噬菌体VCSM13超感染,使P97基因随机片段以融合蛋白的形式展示于噬菌体表面,从而成功构建了P97基因特异性噬菌体随机肽库。用PCR及DNA测序法鉴定所建文库的随机性和多样性,测定肽库的滴度并计算库容量。结果:所建肽库的容量约为1.8×104,滴度约为1.3×1012TU/mL,PCR检测及DNA测序结果显示插入片段具有随机性和多样性。结论:所建随机肽库具有较好的随机性和多样性,能够满足后续的抗原表位筛选,为进一步的深入研究奠定了基础。

Objective：To construct a phage gene-targeted random peptide library of C-terminal of Mhp P97 gene for P97 epitope screening. Method ： In this study, Strain Z of Mhp was incubated with A26 liquid cuhure medium, the gehome DNA of Mhp was extacted and purified, then partial C-terminal sequences of P97 gene were amplified by PCR. The PCR products were digested with DNaseI and the random fragments between 50 bp N 100 bp were recovered. The random fragments （50 bp- 100 bp） were cloned into the linear vector pC89 phagemide and the recombinant phagemides were used to transform competent E. coli XL1-Blue. The transformed cells were infected by helper phage VCSM13 and the recombinant phagemids were rescued. The fusion proteins were displayed on the surfaces of the recombinant phages. A phage gene-targeted random peptide library of P97 gene of Mhp was constructed, which was carefully checked with PCR and DNA sequencing for the randomicity and diversity. The capacity and liter of the peptide library was calculated. Result： The capacity and liter of the peptide library was calculated as 1.8 × 10^4 clones and 1.3 × 10^12 TU/mL; PCR analysis and DNA sequencing of inserted fragments showed the randomicity and diversity of the constructed library. Conclusion： The randomicity and diversity of the phage gene-targeted random peptide library is proved good enough for application in screening of P97 epitope.