Overexpression of thymidylate synthase in colon bacillus

Abstract

The invention relates to a new method of overexpression of thymidylate synthase in colon bacillus. TS (Thymidylate Synthetase) is a necessary enzyme for synthetizing catalytic thymidylate in vivo, repression of TS activity in rapidly proliferating cells can hander DNA synthesis to cause cell death, TS is an important target for cancer chemotherapy for the past many years, and research on enzymatic properties thereof contributes to the development of new anti-cancer drugs. Human TS gene is eukaryotic gene and contains a large number of prokaryote rare codons, and pure enzyme is less and the operating volume is large with the traditional expression method. According to the invention, colon bacillus expression plasmids TS-pET-28b(+) containing wild type human thymidylate synthase gene is constructed by adopting a molecular biology method, transferred to a bacterial strain Rosetta (DE3) containing the eukaryote rare codons, and are efficiently expressed through optimization of fermentation conditions, thus, the system has the characteristics of high expression level, low cost, and the like.

Description

Technical field: [0001] The present invention relates to the cloning of the wild-type human thynidylate synthase (thynidylate synthase, TS) gene without site-directed mutation, the construction of the prokaryotic expression vector TS-pET-28b(+) containing the gene, and the expression of the gene in High expression in E. coli. technical background: [0002] With the rapid development of the world economy, people are more and more exposed to a series of carcinogens including tobacco, alcohol, viruses and infections, radiation and radiation, and there are about 11 million new cases worldwide every year. The incidence of cancer in China accounts for 20.3% of the world's total, with more than 2.2 million new cases each year. Cancer has become a major burden of disease in the world, and the global incidence is also on the rise. Developing a high-efficiency, low-toxicity anti-cancer drug has become a dream pursued by governments and scientific researchers. However, the low efficie...

AI Technical Summary

Problems solved by technology

However, gene sequence analysis found that the coding sequence of human TS gene cDNA contains a large number of rare prokaryotic codons, accounting for about 10.09% of the total number of TS gene coding sequences (see Table 1 for details). The eukaryotic codons in the sequence are mutated one by one to the commonly used codons in prokaryotes, which can achieve high-efficiency expression of TS protein, but the amount of experimental operation is huge, the difficulty is very high, and the economical applicability is poor

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