Takehiro Kokuho

Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-N-Acetylglucosaminidase Inhibition

We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-typeN-glycans, the most frequent structure of insectN-glycan is the paucimannosidic type, Man3GlcNAc2(±Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated β-N-acetylglucosaminidase (GlcNAcase)-mediated removal ofN-acetylglucosamine residues from the precursorN-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion ofN-acetylglucosamine residues from intermediateN-glycans is critical for the assembly of paucimannosidicN-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus.

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 microg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 microg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.

Production of biologically active, heterodimeric porcine interleukin-12 using a monocistronic baculoviral expression system.

A baculoviral expression system for the production of biologically active, heterodimeric interleukin (IL)-12 was developed by utilizing foot-and-mouth disease virus (FMDV) self-cleaving peptide, 2A. Recombinant porcine IL-12 (rpoIL-12) was produced by insect cells after infection with recombinant baculoviruses expressing the gene encoding a fusion protein of p35 and p40 subunits of IL-12 connected with 2A. By reducing and non-reducing SDS-PAGE analyses, it was demonstrated that rpoIL-12 had a heterodimeric structure which was resulted from 2A-dependent cleavage of the precursor fusion protein. In contrast, uncleaved, monomeric rpoIL-12 was produced by infection with baculoviruses expressing the gene lacking the 2A sequence. To assess the biological activities of these recombinants, we performed the proliferation assays of PHA-activated human PBMCs. The heterodimeric rpoIL-12 induced proliferation in a dose-dependent manner, whereas the uncleaved rpoIL-12 did not. Moreover, such biological activity was specifically inhibited by addition of anti-IL-12 antibodies or rpoIL-12 p40. These observations suggest that FMDV 2A can exert its self-cleaving activity even in a heterologous system, and that biologically active, heterodimeric rpoIL-12 can be generated by monocistronic expression of the p35/p40 fusion gene in combination with the 2A sequence.

High-efficient expression of porcine IL-2 with recombinant baculovirus infected silkworm,Bombyx mori

Biologically active porcine Interleukin-2(poIL-2) was produced fromin vitro andin vivo baculovirus expression systems, namely theAutographa californica nuclear polyhedrosis virus (AcNPV)-cell culture system and the Hybrid nuclear polyhedrosis virus (HyNPV)— silkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.

Molecular cloning and expression profile analysis of a novel β‐D‐N‐acetylhexosaminidase of domestic silkworm (Bombyx mori)

Lepidoptera such as the domestic silkworm (Bombyx mori) produce proteins modified with unsialylated, mannose‐rich moieties known as ‘high mannose‐type’N‐glycans. However, we observed that, under intrinsic acetylglucosaminidase (GlcNAcase)‐inhibited conditions, moth cells tend to synthesize different types of glycoform with sialic acid modification. To identify molecules essential to assemble Lepidoptera‐specific N‐glycans, we performed BLAST analysis on the silkworm genetic database and isolated the entire coding sequence of novel Bombyx GlcNAcase, BmGlcNAcase 2. This enzyme showed weak homology to currently known, lysosome‐associated eukaryotic hexosaminidases, but it revealed remarkable similarity with recently reported glycosyl hydrolases of Spodoptera and Bombyx. Interestingly, BmGlcNAcase 2 was found to be expressed in embryos and in certain tissues of molting larvae (i.e. ovary, fat bodies, mid‐intestine, skin), but not in pupae, suggesting its unique function in the carbohydrate metabolism of juvenile silkworm.

Cloning of porcine interleukin (IL)-12 receptor β2 (IL-12Rβ2) gene and its application to a rapid biological assay for human/porcine IL-12

Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.

Effects of the Environmental Improvements and Probiotic Supplement on Productivities of Weaned Pigs

茨城県内の離乳後死亡率の高い農家(母豚 140頭,一貫 経営)を試験対象とした。2010年 10月中に生まれた子豚(n =140)を離乳時の体重に応じて 4群(重,中,軽,極軽) に分け,下記の 4つの処置群(n=35×4群)をランダム に割り振った。 A 豚舎全体を清掃・消毒し(清掃群),飼料に生菌剤 Lactobacillus casei I-5(1%)を添加 B 豚舎全体を清掃・消毒し(清掃群),通常の飼料を 投与 C 従来通りの豚舎で(従来群),飼料に生菌剤(1%) を添加 D 従来通りの豚舎で(従来群),通常の飼料を投与 対象豚の健康状態を観察し,体重を 1週毎に離乳後 9週 齢まで測定した。また唾液を離乳直後,離乳舎移動後 1週 間,同 4週間および肥育舎移動(離乳舎移動後 6-7週間後) 直後に個体ごとに採取し,唾液中のストレス指標(コルチ ゾール)を測定した。清掃群,従来群の畜舎内のメタン, アンモニア,二酸化炭素,一酸化二窒素,エアロゾル,空 中浮遊一般細菌を継続的に測定した。以上の結果を清掃群・ 従来群,生菌剤投与群・非投与群,性別(去勢雄・雌), 離乳時体重などの要因に分けて解析した。

Evaluation of the Environmental Factors, Stress Markers and Economical Losses in a Swine Farm with High Mortality Rate

茨城県内の1養豚農家においては，呼吸器病や下痢による高い死亡率による損耗が大きな問題となっていた。当農家では，木造開放式の比較的環境の良い離乳期育成舎（以下育成舎A）と，糞尿処理能力が十分でないコンクリート製の離乳期育成舎（以下育成舎B）を有していた。今回当該農家の育成豚の追跡調査を行い，異なる育成舎の空気環境やそこで飼養される育成豚にかかるストレスや生産性について調査を行い，得られた結果より死亡事故による経済的損失の算出を試みた。