猪肺炎支原体P97基因特异性随机肽库的构建与初步筛选

Construction and Screening of Phage Gene-targeted Random Peptides Library of P97 Gene of Mycoplasma hyopneumoniae

构建猪肺炎支原体(Mhp)P97基因特异性随机肽库,探索从中筛选Mhp抗原表位的可行性。PCR扩增获得Mhp P97基因的部分C-端序列,产物用DNaseⅠ消化后将回收的P97随机片段(50 bp^100bp)克隆到pC89pⅧ型噬菌粒载体中,转化XL1-blue感受态细胞,辅助噬菌体VCSM13超感染,获得MhpP97基因特异性噬菌体随机肽库。经过三轮亲和筛选,有效地富集了噬菌体阳性克隆。结果显示,所建肽库的容量约为1.8×104,滴度约为1.3×1012pfu/mL。随机挑取10个阳性克隆进行ELISA检测,结果显示除1号孔外均为阳性。

The objective of this study was to construct a phage gene-targeted random peptides library of Mhp P97 gene and to investigate the feasibility of screening epitops of Mhp.The partial C-terminal sequences of Mhp P97 gene were amplified by PCR.The PCR products were digested with DNaseI,then the recovered fragments（50 bp-100 bp） were cloned into the properly handled pC89 phagemids and the recombinant phagemids were used to transform competent E.coli XL1-blue.The transformed cells were infected with helper phage VCSM13,and the recombinant phagemids were rescued,then the phage gene-targeted random peptide library of Mhp P97 gene was constructed.The library was screened by affinity reaction,and the positive clones were concentrated.The results are as follow： The capacity and liter of the peptide library was calculated as 1.8×10^4 clones and 1.3×10^12 pfu/mL.The 10 positive clones got from the library randomly were tested by ELISA,all of them showed positive results except the number 1 hole.