【摘要】 本文报告了美洲鲽抗冻蛋白基因的克隆及在E.coli中表达的研究,以质粒P^CT5作为抗冻蛋白基因的供体,p^ORF-2作为表达载体。用HpaⅡ酶从质粒p^CT5上切下抗冻蛋白基因片段,再经Bal 31酶,绿豆核酸酶处理,连接上BglⅡ接头,然后插入到p^ORF-2的BglⅡ位点上,借助于p^ORF-2上的β-半乳糖苷酶基因的活性,使含正确插入抗冻蛋白基因的克隆呈现出蓝色菌落,共获得4000多个转化子,其中有201个蓝色克隆。对于50个蓝色克隆提取质粒DNA,电泳后发现均大于p^ORF-2。用BglⅡ消化后,可以发现有300—1500bpDNA片段,同时确定了抗冻蛋白基因在p^ORF-2中的插入方向,对于正确插入的克隆作出部分限制性内切酶图谱,测定出插入的抗冻蛋白基因片段的DNA序列,然后将重组质粒从E.coli MH1000菌株转化到E.coliTK1046中,研究分析表达产物,SDS—聚丙烯酰胺凝胶电泳结果证明插入的抗冻蛋白的基因已表达,有明显的融合蛋白带,分子量大于β-半乳糖苷酶、是由大肠杆菌的外膜蛋白F,抗冻蛋白和β-半乳糖苷酶组成。融合蛋白含量占总蛋白的20％左右。更多还原

【Abstract】 The present paper describes a cl皀ing of antifreezing protein gene of winter flounder (Pseudopleuronectts americanus) and its expression in E.coli:The AF gene was isolated from pCT15 containing a 326 bp cDNA of AF gene in PstI site and a 477 bp fragment in Hpall site. The fragment to be cloned was isolated by Hpall digestion but PstI (two PstI sites within AF gene) and shortened by Bgl 31, then a Bglll linker was added after treatment of Mung Bean Nuclease. The treated fragment were inserted into pORF-2, a expressing vector, in BalII site and transtorrned into E. coli MH1000. The 201 blue colonies out of 4000 colonies were screened by inicator plate containing IPTG and X-gal and by in situ hybridazation. The DNA of 50 recombin-ants were extracted for further analsis. By Bglll digestion and agarose electr- ophoresis, a range of inserts from 300-1500by were identified. The direction of inserted gene was determindcd by analysis of HinfI fragment in size ard the CIaI fragment of pORF-AF was mapped and the Bg1II fragment of pORF-AF was sequenced. For gene expression, the pORF-AF was transformed into E. coli TK1460 and the productss subject to SDS-polyacylamide gel electrophor-esis. An extra band could be seen clearly as compared with control. The fusion protein is about 100KD and more than β-galactosidease in molecular wcigth. The fusion protein is about 20% in total proteins.We Thank Dr. D., Curries and all of friend in The Department of Biotechnology, Alberta Research Council, who gave us lot of help. This work was supported by TRT of Alberta, Canada.更多还原