霍乱毒素B亚基基因上游序列对B亚基表达水平的影响

The Upstream Sequence of Cholera Toxin B Subunit Gene: Effect on CTB Expression

本研究通过缺失突变和移码突变研究了ｃｔｘＢ基因上游Ａ基因部分序列对ｃｔｘＢ表达水平的影响，结果表明：（１）将霍乱毒素操纵子ＸｂａＩ－ＥｃｏＲＩ片段克隆至ｐＵＣ１９，构建的质粒ｐＵＣ１９ＣＴＢ中Ａ亚基的部分序列不能翻译，该质粒转化大肠杆菌后ＣＴＢ的表达产量为３０μｇ／μｌ；（２）在质粒ｐＵＣ１９ＣＴＢ的ＸｂａＩ位点引入移码突变，构建质位ｐＭＣ０２Ｃ，使Ａ亚基基因部分序列能够翻译至自然的终止密码，Ｂ基因的表达水平却降低一倍；（３）质粒ｐＵＣ１９ＣＴＢ缺失ＸｂａＩ－ＣｌａＩ（５５０ｂｐ）的非翻译序列，构建质粒ｐＭＣ０３（Ａ亚基序列不翻译），该质粒转化大肠杆菌后ｃｔｘＢ基因的表达水平降低１倍；（４）在质粒ｐＭＣ０３中ｃｔｘＢ基因上游引入移码突变，构建质粒ｐＭＣ０３Ｃ，其Ａ亚基部分序列能够表达，这时ＣＴＢ的表达水平较ｐＭＣ０３低得多，较ｐＵＣ１９ＣＴＢ低２０倍以上。进一步研究表明，在ＸｂａＩ－ＣｌａＩ片段之间存在具有较强启动子活性的ＤＮＡ序列是５５０ｂｐ非翻译序列缺失后ＣＴＢ表达水平降低的原因。对Ａ基因的阅读框架变化影响ＣＴＢ表达水平的可能机理进行了讨论。

In this work, we have studied the effect of cholera toxin A structure gene on the expression of the distal ctxB gene by the methods of deletion and frame-shift mutation. The results showed that: The expression level of Plasmid pUC19CTB,which was constructed by cloning the XbaI-EcoRI restriction fragment into pUC19 and ctxA gene was out-frame with lacZ' gene, is about 30μg/ml; If a frame shift mutation was introduced at XbaI site of pUC19CTB so that the cholera toxin A ge ne was inframe with lacZ' and could be translate, the expression level of ctxB was decreased to 12μg/ml, When A further deletion from XbaI to ClaI of cholera toxin A gene(about 550bp)was made and ctxA was outframe with LacZ', ctxB expression was decreased two fold compared to pUC19CTB, If the ctxA was inframe with LacZ' so ctxA gene could be translated, the expression level of CTB is much lower than the plasmid outframe with lacZ'. These observations could not be explained by the current knowedge about genetical regulation of cholera toxin operon.The promoter we found located in the cholera toxin A subunit gene, which is responsible for the expression of cholera toxin B subunit, may answer the question why the 550bp non coding sequence could enhance the expression of cholera toxin B subunit.