鲜味八肽的表达载体构建及表达效果验证

鲜味肽是近年来新发现的呈味物质,但国内外对其呈味效果存在争议。导致这一争议的可能原因是验证鲜味肽呈味效果的方法主要是采用化学合成法。为了制备生物源鲜味肽,以便对鲜味肽呈味效果进行再评价,本文以争议性鲜味八肽为目标肽,采用基因工程法,构建了制备生物源鲜味肽的表达载体,并对其表达效果进行了验证。根据争议性鲜味八肽的一级结构Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala和大肠杆菌E.coli BL21(DE3)的密码子偏爱性,设计了该鲜味八肽的DNA序列,并将其克隆在pET-32a(+)上形成表达载体。通过菌落PCR检测阳性克隆、重组质粒鉴定及工程菌的诱导表达,结果发现,鲜味八肽的目的基因成功重组在pET-32a载体上,所得pET-32a鲜味肽重组载体转化大肠杆菌后能够正常表达融合蛋白。这为后续获得生物源鲜味八肽奠定了基础。

Construction and Validation of an Expression Vector for Umami Octapeptide

Umami peptide is a recently identified umami substance, but its taste is controversial domestically and internationally. The possible reason for the controversy is that the method of verifying its taste is mainly based on chemical synthesis. In order to obtain natural umami peptide for the further evaluation of the umami peptide taste, the controversial umami octapeptide was used as the target peptide, gene engineering was adopted to construct an expression vector for the octapeptide, and its expression effect was verified. The DNA sequence of the octapeptide was designed according to the primary structure of the octapeptide, Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala, and codon bias of Escherichia coli BL 21 (DE3), and was cloned into pET-32a (+) to form the expression vector. Through colony polymerase chain reaction (PCR) identification of positive clones, identification of the recombinant plasmid, and the induced expression of the engineered bacteria, it was found that the target gene of the octapeptide was successfully restructured into the pET-32a vector, and the recombinant pET-32a vector-transformed E. coli could normally express the fusion protein. This method will lay a solid foundation for obtaining the natural octapeptide in the future.