摘要
目的探讨蛋白表达技术在支原体研究中的作用。方法以解脲脲原体（ｕｒｅａｐｌａｓｍａｕｒｅａｌｙｔｉｃｕｍＵＵ）为例，根据所研究ＵＵ的目的ＤＮＡ片段设计一对特异性引物，经ＰＣＲ扩增得到目的ＤＮＡ，克隆后运用表达质粒载体ｐＧＥＸ２Ｔ在ＩＰＴＧ诱导下成功诱导了蛋白的表达，并用Ｗｅｓｔｅｒｎ印迹法检测其抗原活性。结果所表达的蛋白具有很好的抗原活性。结论蛋白表达应用于支原体研究中，为研究支原体的致病性与种类或型别的关系提供了重要的手段。实验中我们体会到：（１）在设计引物时，要注意避开ＵＧＡ，可用ＵＧＧ代替。（２）ＩＰＴＧ的浓度从０．１ｍｍｏｌ／Ｌ～１ｍｍｏｌ／Ｌ均有报道，我们实验证实两种浓度在诱导蛋白表达中无差别，因而推荐使用０．１ｍｍｏｌ／Ｌ。（３）蛋白诱导的时间以４～４．５ｈ为最佳。
Objective To explore the role of protein expression's technique in mycoplasma research.Method A couple of specific primers were designed according to objective DNA fragmants of UU.The target fragment was amplified and cloned.Protein expession was induced successfully with IPTG,and its antigen activity was detected by western blotting.Results The expression protein has good antigen activity.Conclusion It provided important tools to study pathogenic relation with kinds or serovars of mycoplasma.The authors considered:(1)To design primers attention must be paid to evade UGA and to take place of UGG.(2)Two consistency,i.e.0.1and 1 mmol/L of IPTG had no difference in our experiments,so 0.1mmol/L of IPTG is judicious.(3)The best time of protein expression is 4～4.5 hours.
出处
《中国皮肤性病学杂志》
CAS
北大核心
1999年第4期195-196,共2页
The Chinese Journal of Dermatovenereology
