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ABSTRACT. We developed a procedure for the large-scale purification of bovine interferon-τ (boIFN-τ) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-τ (rboIFN-τ) was efficiently produced in the silkworm infected with boIFN-τ cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 × 10 8 U/mg) of 91% pure rboIFN-τ by means of a combination of the ANT, followed by QSC and CSCC. KEY WORDS: baculovirus, bovine, IFN-τ, purification, silkworm.J. Vet. Med. Sci. 66(11): 1395-1401, 2004 Interferon (IFN)-τ, originally named trophoblast protein-I, is the major secretory protein of trophoblastic tissues and plays an important role in the maternal-fetal recognition of pregnancy in ruminants [1,8,19]. Interferon-τ has been classified as a member of the large multi-gene type I IFN family, and has about 85 and 70% overall homology to IFN-ω on the bases in the cDNA nucleotide and amino acid sequences, respectively [4,9,20]. As expected from the homology, IFN-τ possesses most of the major functions of IFNs, including anti-viral activity, but does not lead to a febriferous reaction, a serious side effect of the IFN therapy. Because of its biological activities, IFN-τ is applied as a useful tool in ongoing studies on the establishment of pregnancy, and is also expected to be used as an agent to improve the pregnancy rate through the control of implantation and to treat viral diseases. For these applications, it is essential to establish a mass production procedure for purified IFN-τ. Up to now, recombinant ovine and bovine IFN-τ boIFN-τ) had been expressed by baculovirus, yeast and Escherichia coli (E.coli) gene expression systems [3,6,16]. Among these, the baculovirus gene expression system is the most suitable system for producing recombinant IFN-τ for animal experiments and practical biological agents because of its similarity in terms of post-translational modifications, such as glycosylation, phosphorylation, conformation, antigenicity and so on [14]. As the baculovirus gene expression system, Autographa californica (Ac) nuclear polyhedrosis virus (NPV) with insect culture cells as the host and Bombyx mori (Bm) NPV with silkworm larvae as the host were popularly used. The advantage of the AcNPV-insect cell culture system is the absence of serum protein contamination in the culture fluids, since the cells can be cultured wit...

ABSTRACT-Human chymase is a mast cell-derived serine proteinase, which is a non-angiotensin converting enzyme angiotensin II-generating enzyme. It appears to participate in various diseases, but it is unclear whether chymase plays major roles in physiological and pathophysiological functions in vivo. To obtain information on the physiological and pathophysiological functions of chymase and to search for diseases in which chymase participates, in the present study, we aimed at producing recombinant human chymase in large quantities and at developing an ELISA system using anti-human chymase antibodies. A recombinant human chymase was produced by a silkworm-baculovirus expression system. The recombinant chymase in active form was efficiently purified from larval hemolymph using cation-exchange and heparin column chromatography. This recombinant enzyme was enzymatically identical with native human chymase. On the other hand, the stability of the recombinant enzyme in cultured medium for mammalian cells at 37°C was very high as compared with the stability of the native enzyme; 20% of the activity was maintained 120 h after addition of medium. These results indicated that the recombinant enzyme could also utilize in vitro and in vivo assay systems. We obtained several anti-chymase monoclonal antibodies by using the recombinant human chymase as antigen. These antibodies were used to construct an ELISA system for measuring the chymase concentration in blood. As a result of preliminary examination using this ELISA system, it was shown that the chymase concentration in each serum from hypertensive patients is significantly higher than in normal serum. The ELISA system will be applicable for clinical diagnosis and in vivo evaluation systems for chymase-targeting drugs.

We previously demonstrated the biological activities of single-chain recombinant gonadotropins (scGTHs) of goldfish Carrassius auratus follicle-stimulating hormone (scFSH) and luteinizing hormone (scLH), produced by a baculovirus-silkworm larvae system, by using in vivo bioassays with some fishes including Japanese eel Anguilla japonica. Among the bioassays, we succeeded in induction of spermatogenesis of sexually immature male Japanese eels by both scFSH and scLH, especially resulting in the occurrence of spermatozoa in scLH-administered males. However, those recombinant hormones did not induce enlargement of testes. In order to further confirm the potency of recombinant GTHs for use in aquaculture species, we administered scFSH and scLH to males of Japanese eel at higher dosage and frequency (eight times with 2-5 days interval) than those of the previous study (five or six times with 7 days intervals), including combination of scFSH and scLH administration (scFSH-scLH). Gonadosomatic indices (GSI) of scLH-and scFSH-scLH-administered males were larger than those of initial control males and of control males that were injected with saline. Enlargement of testes was also confirmed by measurement of testicular lobe size in scFSH-, scLH-, and scFSH-scLH-administered males. By histological observation, occurrence of spermatozoa was confirmed in scLH-and scFSH-scLH-administered eels. Although milt production was not induced, higher dosage and frequency of scGTH administration was effective in promoting testicular development of immature eels. Thus, single-chain fish GTHs produced by the baculovirus-silkworm larvae system could be a useful tool for promotion of gonadal maturation in aquaculture fishes.

While the Baculovirus Expression Vector System (BEVS) mainly uses insect cell lines, such as Sf9 cells, the robust high expression system using silkworm has also been developed. We have further improved technologies for enhancement of virus recombination, reduction of proteolytic degradation and aggregation, and more reliable promoters. These developments made it possible to achieve high and soluble expression of recombinant proteins. We review here such technology developments, advantage of using silkworm and some example applications. There are areas where this technology can be further improved as implicated in the end.