| The production of proteinic medicine by gene engineering is one of the international investigative hot spots at present. It is also a problem for people to exploring 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. In order to determining the feasibility of producing heterologous proteins by animal mammary glands as a temporary expression system, we chose two kinds of target proteins, one is the human protein C (hPC) which is a medicinal protein exist in plasma, another is the human nerve growth factor beta (hNGF-β) which is a cytokine used for curing degenerative diseases. We found that the cDNA genes of the two kinds of proteins mediated by recombinant adenoviruses could be expressed efficiently in rabbits'mammary glands. And the functional target proteins had been obtained from milk of rabbits. 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 total RNAs were distilled from human embryo heart and liver, the cDNAs of hPC and hNGF-βwere obtained from the two total RNAs by RT-PCR, respectively. The correctness of the two cDNAs was confirmed by analysis of the nucleotide sequences and by compares of their amino acid sequences with the published amino acid sequences. (2)The eukaryotic expression vectors were constructed and the heterologous genes were expressed in cultured cells. It was confirmed that the cDNAs of hPC and hNGF-βconstructed in expression vectors could be used for expression. (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 genes of heterologous proteins in recombinant adenoviruses were expressed in cultured mammary gland epithelia. (6)The recombinant adenoviruses were injected into the mammary glands of rabbits for infecting the mammary glands epithelia. The rhPC and rhNGF-βwere examined from the milk of rabbits, respectively, and the functions of the two proteins were confirmed by analyzing their activity. (7) The rhPCs which expressed in milk of rabbits by the two kinds of vectors, AdPG and AdPF, were intercompared. The result would provide a basis for perfecting this technical system by optimizing the vector.The main results are as follows:(1) The hPC and hNGF-βcDNA were successfully amplified by RT-PCR from a human embryo heart and liver tissues, respectively. The cDNA sequences of hPC and hNGF-βwere in accordance with the sequences in Genbank by DNA sequence analysis. The amino acid sequences coding by the two cDNAs were also in accordance with the sequences of the two proteins which had been published.(2) Four expression vectors were constructed on base of pcDNA3.1+, pIRES and pShuttle-CMV vectors, p3PG, p3NG, pShPG and pShNG. The hPC was constructed into p3PG and pShPG and the hNGF-βwas constructed into p3NG and pShNG. The neomycin resistance gene (Neor), which permits 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 p3PG and p3NG. The GFP gene was also constructed into pShPG and pShNG served as a live marker. Thus construction facilitated checking the express of target gene and screening out the transformed cells.(3) The shuttle vector pShPF including hPC gene and furin gene was constructed successfully.(4) The expressive possibility of these vectors in animal cells was identified by expressions of these vectors in CHO and HEK293 cells. The expressions of hPC and hNGF-βcDNA were investigated. A foundation could be established by this way for following construction of adenoviral vectors containing hPC or hNGF-β.(5) The identified pShNG, pShPG and pShPF vectors were linearized by Pme I, then the linearized vectors were co-transformed E. Coli. BJ5183 with Adeasy plasmid or were directly transformed E. Coli. BJ5183 contained with Adeasy plasmid. The recombinant adenoviral vectors, AdPG, AdPF and AdNG, were obtained by homologous recombination. The directly transforming of pShNG, pShPG and pShPF into E. Coli. BJ5183 contained with Adeasy plasmid validated that the"two step transforming"greatly enhanced the efficiency of homologous recombination.(6) The recombinant adenoviruses, AdPG, AdPF and AdNG, were successfully packaged and propagated in HEK293 cells. These recombinant adenoviruses could infect mammary gland epithelia and the target protein could be expressed in epithelia. (7) High expression level of rhPC or rhNGF-βwas obtained in milk of rabbits by the test of infecting mammary epithelia with recombinant adenoviruses. The highest expression level of rhNGF-βattained 346μg/mL. The highest expression level of rhPC attained 958μg/mL. It confirmed the feasibility of expressing heterologous protein in mammary gland mediated by recombinant adenovirus as vectors.(8) The result of compare of the two kinds of vectors constructed with hPC cDNA showed that the furin could facilitate the expression of the mature protein of rhPC. But the effect was not so much as that of reported as before. The reason may be that the difference of the constructed vectors made the difference in expression of furin, and at last the rhPC zymogen could not be degraded totally into mature rhPC.High expression level of rhPC and rhNGF-βwere obtained in milk of rabbits by infecting mammary epithelia with recombinant adenoviruses. The gene of furin was constructed in down-steam of the hPC gene in adenoviral vector and it could facilitate the expression of mature protein of rhPC. 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 or in the field of producing gene engineering medicines. It opened a new way for producing transgenic medicinal proteins.
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