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目的 建立以pyrG基因为选择标志 ,以黑曲霉菌为宿主菌的蛋白质表达系统。 方法 以PCR扩增获得的黑曲霉菌 pyrG基因的部分序列片段为探针作两次噬菌斑原位杂交 ,从黑曲霉菌ATCC 12 0 49基因文库中克隆获得长度为 9.8kb的DNA片段 ,将其中含 pyrG基因的 2 .3kb片段亚克隆至表达性载体PIGF中 ,获得重组质粒。对克隆基因进行了全基因测定和分析。以黑曲霉菌ATCC 12 0 49的 pyrG缺陷株M 5 4为受体菌 ,用聚乙二醇 /氯化钙方法作基因转化实验 ,将重组质粒导入该受体菌并在其中表达。结果 从黑曲霉菌ATCC 12 0 49基因文库中克隆获得了pyrG基因 ,构建了含该基因的重组表达性载体 pYG1.2。序列分析表明该基因与黑曲霉菌L112的 pyrG基因编码序列的同源性为 93 .9% ,两者经推导的氨基酸的同源性为 98.9%。重组质粒 pYG1.2可使黑曲霉菌ATCC 12 0 49的 pyrG缺陷株M5 4发生基因转化 ,成为Pyr+ 。结论 在克隆了 pyrG基因的基础上成功构建了以 pyrG基因为选择标志 ,以黑曲霉菌为宿主菌的重组表达性质粒
Objective To establish a protein expression system with pyrG gene as the selectable marker and Aspergillus niger as host. Methods A partial DNA fragment of pyrG gene of Aspergillus niger obtained by PCR amplification was used as a probe for two-stage plaque in situ hybridization. A DNA fragment of 9.8 kb in length was cloned from the Aspergillus niger ATCC 12 0 49 gene library. The 2.3 kb fragment containing the pyrG gene was subcloned into the expression vector PIGF to obtain a recombinant plasmid. Whole-genome cloning and analysis of genes. Using the pyrG-deficient strain M 5 4 of Aspergillus niger ATCC 12 0 49 as the recipient, the gene was transformed into the recipient strain by the polyethylene glycol / calcium chloride method and the recombinant plasmid was introduced into the recipient strain and expressed therein. Results The pyrG gene was cloned from the Aspergillus niger ATCC 12 0 49 gene library and the recombinant expression vector pYG1.2 containing the gene was constructed. Sequence analysis showed that the homology of this gene to the coding sequence of the pyrG gene of Aspergillus niger L112 was 93.9%, and the homology of the two deduced amino acids was 98.9%. The recombinant plasmid pYG1.2 can transform the pyrG deficiency M5 4 of Aspergillus niger ATCC12 0 49 into Pyr +. Conclusion The cloned pyrG gene was successfully constructed based on the pyrG gene as the selectable marker, Aspergillus niger as the host bacteria recombinant expression plasmid