Method for efficiently expressing and secreting human growth hormone by utilizing escherichia coli

Abstract

The invention discloses a method for efficiently expressing and secreting human growth hormone by utilizing escherichia coli. The recombinant human growth hormone amino acid sequence was digested by double enzymes and inserted into the corresponding digestion site of the vector pET28a(+) to obtain the pET28a-ST II-hGH recombinant vector. Engineering bacteria: escherichia coli containing the pET28aST II -hGH recombinant expression vector. Expression of human growth hormone protein: culturing and inducing the escherichia coli containing the pET28aST II -hGH recombinant expression vector, and collecting the thallus expressing the human growth hormone protein. Purification of recombinant human growth hormone: the thallus are processed by breaking, ultrafiltration and chromatography, ribosome binding site is added to avoid the use of expensive restriction endonuclease Nco I, redundant sequences on the pET28a (+) are removed, the original T7 promoter is used to start the expression of the target gene. efficient secretory expression of the human growth hormone in escherichia coli is realized by using a conventional vector, and the expression quantity of the growth hormone is improved.

Description

technical field [0001] The invention relates to a method for human growth hormone, more specifically, relates to a method for efficiently expressing and secreting human growth hormone by using Escherichia coli, and belongs to the technical field of genetic engineering. Background technique [0002] Human Growth Hormone (hGH) is a single peptide chain, non-glycated, hydrophilic protein hormone secreted by groups of somatotroph cells (α cells) flanking the pituitary gland, containing 191 amino acid residues , the molecular weight is 21700Da, and the isoelectric point is 4.9. Its main function is to stimulate the growth and differentiation of bone and chondrocytes, and regulate the metabolism of protein, sugar and fat. A variety of diseases such as osteoporosis and cardiovascular disease. Growth hormone deficiency is one of the important factors leading to growth disorders in children. Now recombinant human growth hormone has been widely used clinically. [0003] The earlie...

AI Technical Summary

Problems solved by technology

Its disadvantages are: ①Limited sources, scarce yield; ②Low purity, easy to produce antibodies; ③Easy to be contaminated by donor virus, it was banned by the US FDA in 1985

Early 1980s: Genentech developed Met-rhGH, a genetically recombinant human growth hormone containing 192 amino acids, using Escherichia coli (E.Coli) inclusion body technology. Contaminated; ③ not completely consistent with human hGH, easy to produce antibodies, low purity and activity, so it was also eliminated. In the mid-1980s, recombinant human growth hormone containing 191 amino acids was synthesized by common Escherichia coli gene expression technology. However, because its structure is different from that of human pituitary growth hormone, it is still easy to produce antibodies. In addition, its secretion and extraction process is relatively complicated, and it is easy to be polluted or introduce impurities and cause allergic reactions. In the late 1980s, mammalian cells were synthesized by recombinant DNA technology. The recombinant human growth hormone containing 191 amino acids, the product is closer to the natural growth hormone structure, but its disadvantages are: ① high requirements for cell culture, slow reproduction speed, low yield; ② adenovirus contamination - animal source Sexual infection; ③Proliferation-promoting drug pollution—tumor occurrence, in the 1990s, the recombinant human growth hormone synthesized by secretory Escherichia coli gene expression technology, the product was directly secreted outside the bacteria

Its amino acid content, sequence and protein structure are completely consistent with human pituitary growth hormone, and its biological activity, potency, purity and absorption rate are extremely high, which ensures the safety, effectiveness and stability of the product while minimizing the cost of treatment. However, at present, the secretory expression of E. coli has the disadvantages of low expression level, complicated operation, and some ultra-low temperature expression requires high equipment and equipment. In recent years, yeast has also been used to secrete and express hGH, which can express hGH in large quantities, but the yeast expression cycle is long and the medium configuration Complicated, high parameter control requirements and other disadvantages

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