Artificially synthesized pig growth hormone gene and expression and purification method thereof

Abstract

The invention relates to an artificially synthesized pig growth hormone gene and an expression and purification method thereof. In the invention, the DNA sequence of a pig growth hormone PGH suitable for effectively expressing in colon bacillus is designed according to a use principle of the colon bacillus preferred codons, and a PGH protein expressed by the DNA sequence has the same amino acid sequence with the PGH coding region expressed in the pig. The gene is cloned to a pET-28a<+> expression system to transfect the colon bacillus BL21 (DE3). An expression product of the gene detected by electrophoresis accounts for more than 70% of the whole bacterial protein when the induced expression is carried out for 6h at 37 DEG C with the presence of 0.5 mmol / L of IPTG; the product is present in the form of an inclusion body and meets the requirement of large-scale industrial production. The inclusion body is dissolved by using the low-concentration urea (2M) and buffer solution with high pH value (12), and renaturation is carried out through reducing the pH value to 8.5, and the renaturation ratio reaches above 30%. The gel chromatography is carried out for purification to obtain the PGH protein with the purity of 98% or more.

Description

technical field [0001] The invention relates to DNA recombination technology, in particular to a gene encoding animal protein, more specifically to an artificially synthesized porcine growth hormone gene and its expression and purification method. Background technique [0002] my country is a large pig-raising country mainly based on edible pork, with a population of about 500 million heads, and pork output accounts for more than 55% of the world's output. The residues of pig "clenbuterol" have caused many poisoning incidents in our country, which has aroused the attention of our government. Producer-consumer conflicts arise between pork production, quality, and human health. An effective way to solve this contradiction is to find a safer growth regulator, improve the quality and yield of meat products at the same time, so that producers and consumers can accept it at the same time. [0003] Porcine growth hormone (PGH) is a single peptide chain protein hormone (190-191aa)...

AI Technical Summary

Problems solved by technology

So far, most of the PGH obtained by the E. coli expression system exists in the form of inclusion bodies, which require in vitro dissolution of the inclusion bodies and correct protein refolding before they can be active; secondly, the expression level of PGH expression is not high enough, after renaturation , the yield of the purified soluble PGH obtained at last is on the low side, and cannot be used for industrial large-scale preparation. Therefore, the high cost of PGH limits its application in production

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