Mutation alpha-whey protein expression vector, preparation method and application thereof

Abstract

The invention discloses a mutation alpha-whey protein expression vector. A nucleotide sequence of the mutation alpha-whey protein expression vector is shown as SEQ ID NO.1. In the expression vector, a TGT sequence corresponding to No.73 locus cysteine of a gene segment of human-derived alpha-whey protein is mutated into a GCT sequence corresponding to alanine, and meanwhile, ATT, CAG, TCA, AGG and AAC are successively inserted between the No.71 locus amino acid and the No.72 locus amino acid. The invention also discloses a method for preparing HAMLET by utilizing the mutation alpha-whey protein expression vector. The method is used for solving the problems of the present method for preparing HAMLET by utilizing an ion exchange chromatographic column and a metallic nickel chelating column that the production steps are increased, the risk in protein denaturation and degradation is increased and the HAMLET preparation is easy to fail resulted from the cleaning effect of EDTA to NTA metallic nickel chelating column. A quick and large-scale HAMLET preparation method is established.

Description

technical field [0001] The invention belongs to the technical field of biological preparations, and in particular relates to a mutant α-lactalbumin expression vector, a preparation method and an application thereof. Background technique [0002] HAMLET (Humanα-Lactalbumin Made Lethal to Tumor Cells) is a compound with human α-lactalbumin as the main component, which can cause a large number of tumors to die. HAMLET was discovered accidentally in the study of antibacterial effect of human milk, and then proved to be an effector molecule with strong killing effect on tumor cells. In studies using different cell lines, it was found that HAMLET had the effect of selectively inducing apoptosis of transformed cells, some embryonic cells and lymphocytes, but had no significant toxic effect on mature differentiated cells. Further studies have shown that HAMLET has a broad-spectrum anti-tumor effect and can kill more than 40 tumor cell lines from different tissue sources. In animal...

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Problems solved by technology

[0005] The object of the present invention is to provide a kind of mutant α-lactalbumin expression carrier, preparation method and application thereof, solve the existing method for preparing HAMLET by ion-exchange chromatography column and metal nickel chelation column, due to EDTA to NTA The cleaning effect of metal nickel chelate column increases the production steps of mutant α-lactalbumin, and increases the risk of protein denaturation and degradation, which easily leads to the failure of HAMLET preparation

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