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目的:克隆表达平榛(Corh)主要过敏原Corh1的一个片段区基因,并纯化表达的蛋白及检测其免疫学活性。方法:采用生物信息学方法选取Corh1的主要抗原表位区,设计带有酶切位点的特异性引物,采用RT-PCR方法扩增目的基因,将其导入pMD18-T载体中测序。将测序正确的质粒双酶切,并将获得的片段基因导入pET-32a中表达。通过Ni2+亲和层析柱纯化重组蛋白,采用Westernblot方法检测其IgE结合活性。结果:克隆并获得了Corh1的主要表位区基因,基因开放阅读框为243个碱基,编码81个氨基酸,蛋白相对分子质量(Mr)约为9000。表达的蛋白以可溶性为主,纯化出的蛋白有较好的免疫原性。结论:成功地克隆表达了Corh1的主要表位区基因,蛋白具有良好的免疫学活性。
AIM: To clone and express a fragment of Corh1, a major allergen of Corh, and purify the expressed protein and test its immunological activity. Methods: The major epitopes of Corh1 were selected by bioinformatics method. Specific primers with restriction sites were designed. The target gene was amplified by RT-PCR and sequenced. The plasmid with the correct sequencing was double-digested, and the obtained fragment gene was introduced into pET-32a for expression. The recombinant protein was purified by Ni2 + affinity chromatography and its IgE binding activity was detected by Western blot. Results: The major epitope region of Corh1 gene was cloned and sequenced. The open reading frame of the gene is 243 bp and encodes a protein of 81 amino acids. The relative molecular mass of Mr protein is about 9,000. The expressed protein is mainly soluble, and the purified protein has better immunogenicity. Conclusion: The major epitope region of Corh1 was successfully cloned and expressed. The protein has good immunological activity.