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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...

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.