Rabbit-derived paraoxonase 1 mutant and recombinant expression method thereof

Abstract

The invention discloses a rabbit-derived paraoxonase 1 mutant and a recombinant expression method thereof. The method comprises the following steps: cloning and isolating the PON1 gene of a Japanese white rabbit; carrying out genetic engineering modification to obtain a plurality of point mutation genes; carrying out codon optimization on each mutation gene according to the codon preference of escherichia coli, constructing prokaryotic expression vectors carrying different labels, carrying out induced expression, and carrying out screening to obtain Trx-rePON1 and MBP-rePON1 proteins. The mutant can generate complete and active soluble protein in an escherichia coli expression system, has strong hydrolytic performance on para-phenyl acetate and paraoxon-ethyl, and has the hydrolytic activity on the paraoxon-ethyl being respectively 5 times and 19 times of the activity of rabbit-derived background serum PON1 and the activity of recombinant expression PON1 in bombyx mori. According to the method, efficient recombinant expression of rabbit-derived PON1 in escherichia coli can be achieved, and a new choice is provided to application of PON1 in the fields of organophosphorus degradationand biomedicine.

Description

technical field [0001] The present invention relates to a paraoxonase 1 mutant, in particular to a rabbit-derived paraoxonase 1 mutant and its encoding gene and its recombinant expression method in a prokaryotic expression system, belonging to a rabbit-derived paraoxonase 1 mutant and its Recombine the expression domain. Background technique [0002] PON1 (Paraoxonase1) paraoxonase 1, enzymatic classification EC3.1.8.1, belongs to the lactonase family. Since the 1950s, paraoxonase PON1 derived from mammalian serum has been continuously isolated, and PON1 and homologous genes have been cloned from rabbits, mice, cattle and humans (Mackness, M.and B. Mackness, Humanparaoxonase-1 (PON1): Gene structure and expression, promiscuous activities and multiple physiological roles. Gene, 2015.567(1): p.12-21.). Paraoxonase is a natural barrier for mammals, especially humans, to protect and detoxify. It plays a very important role in protecting the body from organophosphate damage. It...

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Problems solved by technology

[0004] The Escherichia coli expression system is simple to operate, has a short cycle, a single genetic background, low cost, and easy purification of expression products; however, for serum paraoxonase PON1 derived from mammals, due to the lack of a protein post-translational modification system in Escherichia coli, both Escherichia coli and The codon preference of mammals varies greatly, so it is difficult to express soluble and active rePON1 protein in E. coli

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