二球悬铃木花粉主要变应原蛋白编码基因克隆的制备

Cloning of gene coding main allergen of Platanus acerifolia pollen

目的:构建含二球悬铃木花粉主要变应原蛋白编码基因的重组质粒。方法:用PCR扩增二球悬铃木花粉主要变应原编码基因的cDNA序列,将扩增的目的片段用双酶切法与质粒pET32a连接,CaCl2法转入工程菌DH5α制备基因克隆。碱裂解法提取质粒DNA,测序鉴定目的片段存在。结果:构建了含二球悬铃木花粉主要变应原编码基因的重组质粒。结论:用天然二球悬铃木花粉可成功地制备含有其主要变应原编码基因的重组质粒,用于变应原蛋白诱导表达。

Objective： To construct a recombinant plasmid including the gene coding the major allergen of Platanus acerifolia pollen. Methods： Total RNA was extracted from natural Platanus acerifolia pollen and cDNA was revese-transcripted. The DNA sequence of major allergen of platanus acerifolia pollen was amplified by polymerase chain reaction （PCR）. The linkage between amplified DNA sequences and plasmid pET32a was realized by double enzyme digestion. The recombinant plasmid was subsequently transferred into E.coli DH5a to prepare gene clone by CaCl2 method. The cloned recombinant plasmid was extracted by alkaline lysis method and the target fragment was confirmed by agarose electrophoresis, followed by PCR and sequencing. Results： The recombinant plasmid including the gene coding the major allergen of Platanus acerifolia pollen was constructed. Conclusion： The recombinant plasmid which included the gene coding the major allergen could be successfully constructed by using natural Platanus acerifolia pollen, and will be available for inducing expression of the recombinant major allergen protein.