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Purification and characterization of an insect haemolymph protein promoting in vitro replication of the Bombyx mori nucleopolyhedrovirus

Chuo-Sanken Laboratory, Katakura Industries Co. Ltd, 1548 Shimo-Okutomi, Sayama, Saitama 350-1332, Japan¹
Laboratory of Molecular Bioengineering, Faculty of Engineering, Mie University, 1515 Kamihama-cho, Tsu, Mie 514-8507, Japan²

Author for correspondence: Toshimichi Kanaya. Fax +81 42 954 2172. e-mail katakura{at}za2.so-net.ne.jp

We have identified a novel protein that promotes Bombyx mori nucleopolyhedrovirus (BmNPV) replication in vitro. This protein was purified from heat-treated haemolymph of B. mori larvae by gel filtration and ion exchange chromatography, and designated as promoting protein (PP). The molecular mass of native PP estimated by column chromatography and that of denatured PP estimated by SDS–PAGE were 9600 Da and 15200 Da, respectively, suggesting that native PP is composed of a single polypeptide and may behave in the column as if it is a smaller protein because of its conformation and/or adsorptive nature. Addition of the PP to the culture medium of SES-BoMo-15A cells derived from B. mori embryos resulted in the strong promotion of BmNPV replication. The promoting activity positively correlated with the amount of PP in the culture medium up to 1 µg/ml, above which maximum virus replication occurred and resulted in the highest budded virus production and polyhedrin promoter-mediated luciferase gene expression of 10000-fold and 6000-fold higher than those without PP, respectively. A cDNA encoding the PP precursor (prePP) was successfully cloned and sequenced. Comparison between the amino acid sequence deduced from the nucleotide sequence of prePP cDNA and the N-terminal 18 amino acids determined for the purified PP indicated that the prePP (154 amino acids) consisted of a mature PP polypeptide (136 amino acids) with a signal sequence at the N terminus. Recombinant PP expressed from the cDNA using a baculovirus vector was similar in molecular mass, immunoreactivity and promoting activity to the native PP.

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