摘要
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.
出处
《动物医学进展》
CSCD
北大核心
2011年第6期5-8,共4页
Progress In Veterinary Medicine
基金
山西省科技厅攻关项目(051056)
关键词
猪肺炎支原体
P97基因
随机肽库
亲和筛选
Mycoplasma hyopneumoniae
P97 gene
random peptide library
affinity screening