| Human growth hormone (hGH), a 191 amino acid single- chain polypeptide is synthesized in the anterior pituitary gland in non-glycosylated form. Its main biological activities are stimulating growth, development, and maintaining the normal functions of the tissues and organs. The hGH has therapeutic applications in the treatment of children with growth hormone deficiency. In addition, applications in the treatment of skin burns, bone fractures, mortification amyotrophy, osteoporosis and bleeding ulcers have been suggested. Owing to the numerous biological functions, the market demands of hGH is enormous. Previously, hGH was extracted from human pituitary gland for clinical application. In this way, the production of hGH was low and the cost was very high. At present, the recombinant hGH is produced by genetic engineering methods. The production of pharmaceutical proteins by gene engineering is one of the international investigative hot spots at present. It is also a problem for people to explore how to produce this kind of medicine efficiently and economically. At present, although there are several expression systems such as bacterial, fungal, insect cells, animal cells, transgenic plants and transgenic animals, all of them have some limitations in any way. The mammary gland has been identified as a suitable alternative to produce biopharmac -eutical and nutritional proteins. The purification process of proteins which are synthesized in the mammary epithelial cells and thereafter secreted into the milk is relatively simple. In order to determin the feasibility of producing heterologous proteins by animal mammary glands as a temporary expression system, we chose hGH as the target protein.We found that the hGH gene mediated by recombinant adenoviruses could be expressed efficiently in the mammary glands of rabbits and goats. The functional target proteins had been obtained from milk.Consequently, it may provide a technical and theoretical basis for exploiting or producing recombinant medicinal proteins by this method.The contents of this study included as follows:(1) The genome DNA was distilled from human pituitary tissue and the hGH gene were obtained by PCR using the genome DNA as template. The correctness of the hGH gene was confirmed by analysis of the nucleotide sequences and by comparing the DNA sequence with the published sequences.(2) The eukaryotic expression vectors were constructed and the heterologous genes were expressed in cultured cells. It was confirmed that the hGH gene constructed in expression vectors could be used for expression. The functions of hGH gene were confirmed by analyzing its activity.(3) The recombinant adenoviral vector was constructed by homologous recombination in bacteria.(4) The packaging of recombinant adenoviral vector was carried out in HEK293 cells, and then the recombinant adenoviruses were amplified in HEK 293 cells.(5)The gene of heterologous protein in recombinant adenoviruse was expressed in cultured mammary gland epithelia.(6)The recombinant adenoviruses were injected into the mammary glands of rabbits and goats for infecting the mammary glands epithelia. The rhGH were examined from the milk of rabbits and goats. The result would provide a basis for perfecting this technical system by optimizing the vector.The main results are as follows:(1) The hGH DNA was successfully amplified by PCR from a human pituitary tissue genome. The DNA sequence of hGH was in accordance with the sequences in Genbank by DNA sequence analysis.(2) The expression vector p3GF which contained the hGH gene was constructed on base of pcDNA3.1+,p3GFP and pIRES vectors. The neomycin resistance gene (Neor), which permited selection of transformed cells and the green fluorescence protein (GFP) gene, which served as a live marker for tracking infected cells or tissues in animal study, were constructed in p3GF. Thus construction facilitated checking the expression of target gene and screening out the transformed cells.(3) The expressive possibility of these vectors in animal cells was identified by expressions of these vectors in CHO cells. The expressions of hGH DNA were investigated. A foundation could be established by this way for following construction of adenoviral vectors containing hGH gene.(4) The shuttle vector pSGF contained hGH gene was constructed correctly.(5) The identified pSGF vector was linearized by Pme I, then the linearized vectors was transformed E. Coli. BJ5183 contained Adeasy plasmid directly. The recombinant adenoviral vecto AdGF was obtained by homologous recombination. The directly transforming of pSGF into E. Coli. BJ5183 contained with Adeasy plasmid validated that the"two step transforming"greatly enhanced the efficiency of homologous recombination. (6) The recombinant adenovirus AdGF was packaged and propagated in HEK 293 cells successfully. These recombinant adenoviruse could infect mammary gland epithelia and the target protein could be expressed in epithelia.(7) High-level expression of rhGH protein was obtained in milk of rabbits and goats by the test of infecting mammary glands with recombinant adenovirus. The highest expression levels of rhGH attained 0.780 mg/mL in milk of rabbits. The highest level of hGH in EGTA-treated goats was 2.35 mg/mL. It confirmed the feasibility of expression heterologous protein in mammary gland mediated by recombinant adenovirus as vector.High-level expression of rhGH protein was obtained in milk of rabbits and goats by infecting mammary epithelia with recombinant adenoviruses in vivo. It confirmed that optimizing the recombinant vector could facilitate the expression of mature heterologous protein in mammary gland mediated by recombinant adenovirus. This method further extended the application of adenoviral vectors. It was the new application in producing transgenic medicinal proteins according to the principle of animal mammary gland bioreactor. This method is important in the field of technical theory of transgene and producing gene engineering medicines. It opened a new way for producing transgenic medicinal proteins.
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