| Human growth hormone (hGH), containing191amino acids, is an important non-glycosylated protein hormone. HGH is correlated with synthesis, storage and secretion of growth hormone in the cells of human pituitary, and it is the major hormone and significant regulating hormone in metabolic pathways in vivo after birth. The physiological functions of hGH mainly includes promoting growth of bone, cartilage and tissue, regulating metabolism, promoting synthesis and lipolysis of protein, regulating immune and maintaining normal operation of body. In the clinical application, hGH can be used to cure growth retardation in children and insufficient growth hormone in adults. HGH is also related to the treatment of cardiovascular disease, the burns, and surgical patients after major surgery. Currently, hGH is used clinically mainly by recombinant DNA technology with the host of E.coli to produce the growth hormone (referred rhGH). However, it involves in purification, denaturing and refolding of recombinant proteins complex. However, the cost may well be very expensive in producing rhGH commercially. Furthermore, hGH has great potency in medicine. It is nevertheless still difficult to be applied on a large scale.The distinguishing feature of the non-pathogenic Gram-positive bacterium Bacillus subtilis is that it produces a dormant endospore called spore under the conditions of nutritional deficiency or other threatening the environment. A mature sopre offers unique resistance properties and can survive in extreme temperature, radiation, and toxic chemicals for hundreds or thousands of years. Therefore, spores become an effective vehicle for delivery of exogenous proteins and bioactive macromolecules. The purpose of this study is to display hGH on the spore surface by surface display technology and construct a nutritional recombinant spore. The main researches and results of this study are as follows:(1) The human growth hormone gene (hgh) was obtained by PCR amplification with the template hgh gene cDNA bought from the Protech corp. Then hgh gene was identified by sequencing and database alignment.(2) Then, the purified hgh gene was cloned into pET-30a to construct recombinant plasmid pET30-hGH, which finally brought a enterokinase site (Asp-Asp-Asp-Asp-Lys) in the front of the hgh gene. (3) To construct fusion expression CotC-hGH integrated recombinant plasmid pJS700-hGH, hgh was cloned into plasmid pJS700and fused with spore capsid protein gene cotC.(4) Plasmid pJS700-hGH was transformed into Bacillus subtilis competent cells. A recombinant Bacillus subtilis strain, which displayed hGH on the spore surface, has been successfully constructed and identified by Western blot and immunofluorescence.(5) Two kinds of silkworm during the5th instar period were inoculated by the oral route with recombinant spores of hGH-displaying.6days later, the results of physiological and biochemical analysis of the silkworm demonstrated significant changes in body weight, pupae weight, body mass conversion efficiency etc. Further Western blot experiments of silkworm blood showed that CotC-hGH fusion protein was precisely cut and processed into small peptide hGH, and hGH protein can be digested and absorbed into the silkworm hemolymph.(6) Groups of mice were inoculated by the oral route with recombinant spores of hGH-displaying.42days later, blood samples were collected to determine serum human growth hormone level. The results indicated that intragastric administration of mice with spores did not cause much gastrointestinal discomfort and oral administration of high dose recombinant spores can increase the serum hGH level in vivo effectively. |
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