FD耐热逆转录酶的部分酶学性质研究

Some Enzymatic Characteristics of FD Thermostable Reverse Transcriptase(FD TRT)

ＦＤ耐热逆转录酶（ＦＤ－ＴＲＴ）来自一株栖热杆菌菌株．通过ＲＴ－ＰＣＲ方法，论文对ＦＤ－ＴＲＴ的部分酶学性质进行了研究．ＦＤ耐热逆转录酶能耐受９５℃的高温，最适反应温度在６５～７５℃左右，这时ＲＮＡ的高级结构能解开，可以提高逆转录反应的效率；同时引物和ＲＮＡ模板识别的专一性强，可大大提高逆转录的特异性．实验表明ＦＤ－ＴＲＴ逆转录反应的最适条件为：２５ｍｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．８，１５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４，１００μｇ／ｍｌ明胶，５００μｍｏｌ／ＬｄＮＴＰｓ，２５ｐｍｏｌ逆转录引物，１ｍｍｏｌ／ＬＭｎＣｌ２，２ＵＦＤ耐热逆转录酶，反应在６５～７０℃下进行．在上述条件下，用ＲＴ－ＰＣＲ可检出少于５ｐｇ外周血总ＲＮＡ中特异的α珠蛋白ｍＲＮＡ．

FD thermostable reverse transcriptase (FD TRT) was isolated from a thermus strain.Some of its enzymatic properties were studied through RT PCR method.FD TRT can endure 95℃,and its optimal reaction temperature is around 65-70℃ when most part of the high structure of RNA are opened,thus it can improve the efficiency of reverse transcription,also as the specificity of recognition between primer and template is increased,it will improve the specificity of reverse transcription.Experimental results show that the optimal reaction condition of FD TRT is as follows:25 mmol/L Tris HCl(pH 8.8),15 mmol/L (NH 4) 2SO 4,100 μg/ml gelatin,500 μmol/L dNTPs,25 pmol reverse transcription primer,1 mmol/L MnCl 2,2 U FD TRT,incubation at 65-70℃.α globin mRNA can be efficiently detected from less than 5 pg total RNA of human peripheral blood cell with RT PCR conducted by FD TRT under the above condition.