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目的克隆腰果主要过敏原Anao2基因,并利用pMAL-c表达载体表达该蛋白。方法提取腰果总RNA,设计特异性引物,反转录-聚合酶链反应(RT-PCR)克隆腰果Anao2基因,将其反转录基因连入pMD18T sim-ple vector,提取质粒、双酶切、鉴定并测序;将测序正确的片段连入原核表达载体pMAL-c,将重组质粒转入BL21宿主表达菌中,丙基-β-D-硫代吡喃半乳糖苷诱导表达目的蛋白Anao2。结果测序结果表明克隆腰果Anao2基因片段全长为1 332 bp,编码443个氨基酸,与GenBank中蛋白序列完全相同;对获得的重组蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定,目的蛋白大小与理论值相符。结论成功克隆并表达了腰果过敏原Anao2。
Objective To clone the Anao2 gene, a major caspian allergen, and use pMAL-c expression vector to express this protein. Methods The total RNA of cashew nuts was extracted and specific primers were designed. Anao2 gene was cloned by reverse transcription - polymerase chain reaction (RT - PCR). The reverse transcript gene was inserted into pMD18T sim - ple vector. Identified and sequenced; the correct sequencing fragment was ligated into prokaryotic expression vector pMAL-c, and the recombinant plasmid was transformed into BL21 host strain. Anao2 was induced by propyl-β-D-thiogalactopyranoside. Results The sequencing results showed that the entire length of Anao2 gene was 1 332 bp, encoding 443 amino acids, which was identical to GenBank. The recombinant protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, The size of the target protein is consistent with the theoretical value. Conclusion The caspian allergen Anao2 was successfully cloned and expressed.