摘要
Ｓｏｕｔｈｅｒｎ印迹结果证明在ＣＦＡ／Ⅱ阳性菌Ｅ４４８１５的诸多质粒中，编码ＣＳ３纤毛抗原的质粒为一约为６０ＭＤ的大质粒；当这些质粒用ＨｉｎｄⅢ消化时，该抗原的编码基因位于５．０ｋｂ的ＤＮＡ片段上。回收该片段并插入到载体质粒ｐＢＲ３２２的ＨｉｎｄⅢ位点，构建了Ｅ．ｃｏｌｉＥ４４８１５株质粒的有限基因库。通过筛选，获得了两种插入方向的阳性重组子。全细胞ＥＬＩＳＡ测定结果表明，只有当插入片段的转录方向和载体质粒中的ｐ１启动子的转录方向一致时，重组质粒才能有效地表达ＣＳ３纤毛抗原；宿主不同时表达水平存在一定差异。ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ印迹试验表明：ＣＳ３纤毛抗原有两种不同的单体形式，分子量大约分别为１５．０ｋＤ和１５．５ｋＤ。ＣＳ３纤毛抗原编码基因的克隆为研究ＣＳ３基因表达调控和研制预防ＥＴＥＣ腹泻疫苗打下了基础。
The
results of Southern blot revealed that the CS3 antigen was coded by a large plasmid(60MD)
isolated fromE44815 strain , and the DNA fragment containing the gene encoding CS3 fimbriae
was about 5.0 kb when the plas-mid was digested by Hind Ⅲ.Limited gene bank was
constructed by recovering all the DNA fragments of about5.0 kb after the plasmid was digested
and inserting it into the Hind Ⅲ site of pBR322.Through screening,we ob-tained two kinds of
positive recombinant plasmids harbouring the cloned fragment in opposite orientation.
Wholecell ELISA demonstrated that only when the orientation of transcription of the cloned
fragment was identical withthat of P1 promoter in the pBR322, could the CS3 antigen be
expressed.And the test also demonstrated that thelevel of expression of CS3 antigen were
different due to different on hosts.SDS-PAGE assay and Western blotidentified that CS3 antigen
had two different forms and the molecular weight were l5 kD and 15.5 kD, respective-ly. Cloning
of the gene encoding CS3 antigen permits us to study the regulation of its expression and to
constructvaccine against ETEC.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
1994年第2期84-88,共5页
Chinese Journal of Microbiology and Immunology
