Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

Muneta, Yoshihiro; Nagaya, Hidekazu; Minagawa, Yu; Enomoto, Chiaki; Matsumoto, Sayaka; Mori, Yoshihide

doi:10.1023/b:bile.0000045643.66758.9d

Cited by 20 publications

(12 citation statements)

References 10 publications

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“…Seven days after the second vaccination, inguinal lymph nodes were excised and the lymphocytes were cultured in 24-well culture plates (5x10 6 per well) with GPC3 peptide-pulsed BM-DCs (1x10 5 per well) in RPMI medium supplemented with 10% horse serum, recombinant human interleukin (IL)-2 (100 units/ml), and 2-mercaptoethanol (50 μmol/l). After culture for 5 days, the cells were recovered and analyzed for their cytotoxic activity against target cells with the TERASCAN VPC system (Minerva Tech) as previously described (15). Briefly, C26, C26/GPC3, RMA-HHD and RMA-HHD-GPC3 cells were used as target cells and labeled with calcein-AM solution for 30 min at 37˚C.…”

Section: Induction Of Gpc3-specific Ctls and Cytotoxicity Assaymentioning

confidence: 99%

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: Preclinical study using mice

Motomura

Ikuta²,

Kuronuma³

et al. 2008

Int J Oncol

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Abstract. We previously reported that glypican-3 (GPC3) is uniquely overexpressed in human hepatocellular carcinoma and melanoma and that it is an ideal tumor antigen for immunotherapy in mouse models. We recently identified both HLA-A24 (A * 2402) and H-2K d -restricted GPC3 298-306 (EYILSLEEL) and HLA-A2 (A * 0201)-restricted GPC3 144-152 (FVGEFFTDV), both of which can induce GPC3-reactive cytotoxic T cells (CTLs). The present study was a preclinical study in a mouse model that was conducted in order to design an optimal schedule for clinical trial of GPC3-derived peptide vaccine. When BALB/c mice were intradermally vaccinated at the base of the tail with K d -restricted GPC3 298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptidespecific CTLs were induced. But the peptide alone could not induce peptide-specific CD8 + T cells. Furthermore, proteomic analyses showed that IFA protected the peptide against degradation in the human serum. Peptide-reactive CTLs were induced by peptide vaccine in a dose-dependent manner. In addition, at least two vaccinations with a single dose >10 μg were needed for the induction of GPC3 298-306 -specific CTLs. But repeated vaccination with a lower dose of GPC3 298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3 144-152 depended on the dose administered. The results of this study suggested that IFA is one of the indispensable adjuvants for peptide-based immunotherapy, and that the immunological effect of peptide vaccines depends on the dose of peptide injected. IntroductionHepatocellular carcinoma (HCC) is one of the most common tumors worldwide, especially in Asian and Western countries (1). Despite advances in diagnosis and treatment, the overall survival of patients with HCC has not significantly improved in the last two decades (2). The effective treatments currently available are only indicated in a relatively small proportion of early stage cases. When patients presents with clinical manifestations of HCC, the tumor is usually advanced, and there are few treatment options. Many HCC patients have type B or C hepatitis or cirrhosis, so patients treated surgically or by other therapies are also at high risk for recurrence. Furthermore, the liver function of such patients is often very poor, so treatment for recurrence is often restricted. As a result, the prognosis of HCC remains poor and new therapies for cancer development and recurrence, i.e., adjuvant therapy, are urgently needed.We previously reported that glypican-3 (GPC3), glycosylphosphatidylinositol (GPI)-anchored membrane protein, is specifically overexpressed in human HCC and melanoma, and that among normal tissues it is slightly expressed in placenta and embryonic liver (3). We found that GPC3 is useful not only as a novel tumor marker, but also as a target antigen for immunotherapy in several studies with mice (4,5). In addition, we identified CTL epitope peptides: HLA-A24-restricted GPC3 298-306 (EYILSLEEL) and HLA-A2-restricted GPC3...

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Section: Induction Of Gpc3-specific Ctls and Cytotoxicity Assaymentioning

confidence: 99%

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: Preclinical study using mice

Motomura

Ikuta²,

Kuronuma³

et al. 2008

Int J Oncol

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“…Insect larvae infected with recombinant baculoviruses can be an extremely useful alternative due to the high levels of protein expression achieved, the lower costs for large-scale production, and a higher efficiency of certain types of posttranslational modifications (19). In the last 10 years, larvae have been successfully used to produce different recombinant proteins (3,5,7,13,16,21,22).…”

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confidence: 99%

Potential Use of the Hemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus Expressed in Rachiplusia nu Larvae as an Immunogen for Chickens

Zoth

Gómez

Carballeda

et al. 2009

Clin Vaccine Immunol

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“…Some proteins have been successfully expressed in silkworm pupae (21)(22)(23)(24). In particular, approx 205 µg of functionally active rPLF was obtained from a single pupa (18).…”

Section: Discussionmentioning

confidence: 99%

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

Chen

Nie

Zhengbing

et al. 2007

Appl Biochem Biotechnol

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Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge. To establish purification methods suitable for mass production, we tried two crude preparation methods: (NH4)2SO4 fractional precipitation and isoelectric precipitation with a combination of gel filtration and ion-exchange chromatography. The isoelectric precipitation method was found to be more efficient. With this method, we eventually obtained approx 11.7 mg of 95% pure product from 1000 g of infected silkworm pupae. The recovery of purified protein was greatly increased, by approx 40%, compared with the other method. The biologic activity of this protein was determined up to 9.0 x 106 colony-forming units/mg in the final purified product.

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Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

Cited by 20 publications

References 10 publications

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: Preclinical study using mice

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: Preclinical study using mice

Potential Use of the Hemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus Expressed in Rachiplusia nu Larvae as an Immunogen for Chickens

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

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