Serine proteinase (SP) and serine protease inhibitor (serpin) gene expression after hormone treatment in the silkworm Bombyx mori (Lepidoptera: Bombycidae)

Serpin (Serine Protease Inhibitor) widely distributes in animals, plants, protozoan, prokaryotes and viruses. Serpin-6 belongs to the superfamily of serine protease inhibitor. In this study, we constructed a prokaryotic vector - pET28a-serpin-6, and used IPTG (isopropyl thiogalactoside) to induce serpin-6 protein expression, then applied the Ni-affinity chromatography to purify the collected recombinant protein. The assay of SDS-PAGE and Anti-his Polyclonal antibody Test showed that the recombinant serpin-6 protein have a molecular weight of 45 kDa. The highly purified sample of the recombinant protein was obtained. The protein has been used as antigen to immunizeKunMing miceusing the 4 times immunization method. We successfully attained the polyclonal antibody of serpin-6.The titer of the antibody is as high as 1:20000, with a good specificity. The polyclonal antibody can provide a practical tool to further study the distribution of protein expression and functions of serpin-6 in different states and breeds ofBombyx mori.

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Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis

Infection and Immunity ◽

10.1128/iai.00866-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5429-5435 ◽

Cited By ~ 43

Author(s):

Shane M. Ceraul ◽

Sheila M. Dreher-Lesnick ◽

Albert Mulenga ◽

M. Sayeedur Rahman ◽

Abdu F. Azad

Keyword(s):

Gene Expression ◽

Salivary Gland ◽

Protease Inhibitor ◽

Serine Protease ◽

Blood Meal ◽

Serine Protease Inhibitor ◽

Dermacentor Variabilis ◽

Mammalian Host ◽

Spotted Fever Group ◽

Kunitz Type

ABSTRACT Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae.

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Growth hormone rapidly activates rat serine protease inhibitor 2.1 gene transcription and induces a DNA-binding activity distinct from those of Stat1, -3, and -4.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.12 ◽

1995 ◽

Vol 15 (1) ◽

pp. 12-18 ◽

Cited By ~ 31

Author(s):

M J Thomas ◽

A M Gronowski ◽

S A Berry ◽

P L Bergad ◽

P Rotwein

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Protease Inhibitor ◽

Protein Binding ◽

Serine Protease ◽

Dna Sequences ◽

Gene Promoter ◽

Serine Protease Inhibitor ◽

Gh Treatment ◽

Gh Receptor

Transcriptional regulation by growth hormone (GH) represents the culmination of signal transduction pathways that are initiated by the cell surface GH receptor and are targeted to the nucleus. Recent studies have demonstrated that the activated GH receptor can stimulate Stat1, a cytoplasmic transcription factor that becomes tyrosine phosphorylated and translocates to the nucleus, where it can interact with specific DNA sequences to modulate gene expression. GH also has been found to induce protein binding to a portion of the rat serine protease inhibitor (Spi) 2.1 gene promoter that is required for GH-induced transcription of Spi 2.1. Using GH-deficient hypophysectomized rats as a model, we show that GH treatment rapidly and potently induces both nuclear Spi 2.1 mRNA expression in the liver and specific nuclear protein binding to a 45-bp segment of the Spi 2.1 gene promoter. A GH-inducible gel-shifted complex appears within 15 min of systemic hormone administration and can be inhibited by an antiphosphotyrosine monoclonal antibody but is not blocked by a polyclonal antiserum to Stat1, Stat3, or Stat4, even though the nucleotide sequence contains two gamma interferon-activated sequence-like elements that could interact with STAT proteins. By Southwestern (DNA-protein) blot analysis, approximately 41- and 35-kDa GH-inducible proteins were detected in hepatic nuclear extracts with the Spi 2.1 DNA probe. Thus, a GH-activated signaling pathway stimulates Spi 2.1 gene expression through a unique mechanism that does not appear to involve known members of the STAT family of transcription factors.

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