E. coli strains TG1 and Rosseta (DE3) were grown at 37˚C in LB medium, pH 7.5, containing 5 g yeast extract, 10 g tryptone peptone, and 10 g NaCl per liter. Recombinant E. coli strains were grown in LB medium with kanamycin (50 μg/mL) and chloromycin (34 μg/mL).

The Qingsong × Haoyue strain of Bombyx mori were reared on fresh mulberry leaves under standard conditions. We isolated the silk gland, head, midgut, epidermis, genital, malpighian tubule, stigma, and fatty body from day 4 and day 5 larvae of the 5th instar. These tissues were washed several times with ice-cold phosphate-buffered saline (PBS, pH 7.4), weighed, and then homogenized in 5 volumes of protein extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitole, 0.5% NP-40, 0.5% sodium deoxycholate) containing protease inhibitor (100 μg/mL phenylmethane sulfonyl-fluoride, 5 μg/mL Aprotinin). Homogenates were centrifuged at 15,000 g for 15 min, and the supernatant was collected for Western blot analysis. Protein concentrations were determined by the Bradford method; bovine serum albumin (BSA; Sigma) was used as a standard [12].

Using our in-house plasmid, TG1-pHelix-Bm-X, as a template, we performed the polymerase chain reaction (PCR) to amplify the Bm-X open reading frame (ORF). We designed a primer pair based on the Bm-X cDNA sequence and introduced restriction endonuclease recognition sites for BamH I and Xho I at the 5’-ends of the primers for subcloning purpose. The sense primer was 5’-CCCGGATCCATGCCGCTGAACAAAACAAC-3’and the antisense primer was 5’-CCGCTCGAGTCAT GACTCGGTTTTATTGTC-3’. The PCR program consisted of a preliminary denaturation step at 94˚C for 5 min, followed by 30 cycles of amplification (denaturation at 94˚C for 30 s, annealing at 55˚C for 30 s, and extension at 72˚C for 1 min), and a final extension at 72˚C for 10 min. PCR products were digested with BamH I and Xho I (Promega), separated by electrophoresis on a 1% agarose gel, and purified by the PCR Rapid Purification Kit (BioDev-Tech). Purified PCR products were subcloned into the expression vector pET-28a (Amersham Biosciences) by T4 DNA ligase (Promega). The expression plasmids containing the PCR products inserted were transformed into competent E. coli (TG1 strain), then picked white colonies. We identified positive colonies that contained the recombinant plasmid among white colonies. Then recombinant plasmids were extracted and digested with BamH I and Xho I, followed by separation and analysis on 1% agarose gel electrophoresis.

Using the BLAST search of Genbank [13] (http://www.ncbi.nlm.nih.gov/BLAST/), we did not find any sequence similarities with the Bm-X sequence. We determined the theoretical molecular weight and isoelectric point by Peptide Mass [14] (http://us.expasy.org/tools/peptide-mass.html). We also performed secondary structure prediction (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html), hydrophobicity prediction (http://www.expasy.org/cgi-bin/protscale.pl), and signal peptide prediction (http://www.cbs.dtu.dk/services/SignalP/) by on-line biological information tools.

The recombinant plasmid pET-28a-Bm-X was transformed into competent E. coli cells [Rosseta (DE3) strain] and the expression of the His-tagged fusion protein with His-tag was induced with 1 mM isopropyl thiogalactoside for 4 h [15]. Recombinant Bm-X (rBm-X) was purified by HiTrap Chelating HP (Amersham), according to the manufacturer’s instructions. The eluted rBm-X was freeze-dried in an alpha 2 - 4 Freeze Dryer (Christ) and then stored at –20˚C.

We injected intraperitoneally a New Zealand White rabbit with a mixture of 1mg purified recombinant Bm-X and Freund’s complete adjuvant (Dingguo). The rabbit was boosted three times with 0.5 mg recombinant Bm-X in Freund’s incomplete adjuvant in one week intervals. Serum was derived from blood collected 10 days after the third injection and purified by HiTrap Protein A HP (Amersham) according to the manufacture’s instructions. After the IgG was filtered through Ultracel PLCHK (Millipore), the purified anti-Bm-X IgG was dissolved in 50% glycerol and stored at –80˚C.

Equal quantities of proteins from various silkworm tissues were separated by SDS-PAGE under reducing conditions, as described by Laemmli (1970). The proteins separated by electrophoresis were then transferred electrophoretically onto polyvinylidene difluoride membranes. Following incubation with purified anti-Bm-X IgG, membranes were washed and incubated with goat antirabbit IgG antibody conjugated to Alexa Fluor 680 (Molecular probe). Finally, membranes were washed three times with Tris-buffered saline plus Triton X-100, twice with double-distilled H₂O, and subsequently scanned by the Odyssey Infrared Imaging System (LI-COR) at 700 nm.

BmN cells were seeded arbitrarily in the dish (Bio-line Instruments) which was used specially for Confocal Microscope. After 12 h, culture medium was removed, and cells were rinsed twice with 1 mL phosphate-buffered saline (PBS), fixed in 3.7% formaldehyde at 25˚C for 10 min. Then the cells were blocked with 3% BSA at 37˚C for 2 h. After that, the cells were incubated with anti-BmX IgG (dilution 1/200) at 4˚C for 12 h (the cells incubated with serum as negative control). After washing three times in PBST (PBS + 0.05% Tween-20, 10 min each), cells were incubated with goat anti-rabbit antibody (dilution, 1/2000, Cy3 labeled, Promega) and 4’-6- Diamidino-2-phenylindole (dilution, 1/2000) at 37˚C for 2 h. Following three times washing with PBST (10 min each), cells were analyzed by Nikon ECLIPSE TE2000- E Confocal Microscope with image analysis software EZ-C1.

Using specific PCR primers on our in-house plasmid TG1-pHelix-Bm-X, we amplified the cDNA of the B. mori-X gene (Bm-X). We have sequenced the Bm-X cDNA and submitted the sequence to GenBank (data on accessing). Sequence analysis revealed that the ORF of Bm-X is 444 bp, and encoded a putative protein containing 147 amino acids. We predicted that the molecular mass of the putative protein was 16.4 kDa (Figure 1(a)). The nucleotide sequence of Bm-X and the deduced amino acid sequence of the protein are shown in Figure 1(a). We also used BLAST to analyse the full ORF of Bm-X and each of the three shorter ORFs starting from three other ATG codons. The result showed that there was no similarity to other species (Figure 1(b)). This indicated that the Bm-X gene is a species-specific gene of Bombyx mori without any previously known homologues.

BLASTX analysis of the translation product deduced from the ORF showed that Bm-X has no significant homology with other proteins in the GenBank database. We conclude that Bm-X is likely a novel species-specific gene and Bm-X protein is important to silkworm. Bioinformatics prediction showed that Bm-X protein contains 12 functional sites (Figure 2), carried with no signal peptide, and has a seconddary structure chiefly consisting of alpha helix and random coil (Figure 3).

The Bm-X was inserted into the expression vector pET-28a with a BamH I site to construct a recombinant plasmid named pET-28a-Bm-X. The recombinant plasmid pET- 28a-Bm-X was then transformed into competent E. coli cells. We confirmed the expression of recombinant Bm-X in E. coli by SDS-PAGE (Figure 4). We estimated that the molecular weight of recombinant Bm-X was 19.96 kDa, which included a 3.56 kDa His-tag. We immunized New Zealand White rabbits with the purified recombinant protein to obtain anti-Bm-X rabbit serum. Using the anti-Bm-X rabbit serum, we detected a strong signal of 19.96 kDa corresponding to recombinant Bm-X in induced E. coli lysate, whereas no signal was detected in the non-induced E. coli lysate (Figure 5).

In order to detect the expression level of Bm-X protein in each tissue, we extracted proteins from the head, midgut, genital, silk gland, epidermis, fatty body, stigma, and Malpighian tubule of the fifth instar larva, and used Western blot analysis to determine the level of Bm-X in each tissue. Immunoblots of these protein extracts re-

vealed that anti-Bm-X serum reacted with a protein over 20.1 kDa in extracts isolated from the midgut, epidermis and silk gland, but did not react with any protein in extracts isolated from other tissues (Figure 6). The amount of the Bm-X protein was the highest in extracts from the epidermis and lower in extracts from the midgut and silk gland.

We used the BmN cells to observe the subcellular localization of endogenous Bm-X protein. Immunostaining showed that Bm-X protein localized in both the cytoplasm and nucleus. But the fluorescence localization signals were stronger in cytoplasm than in nucleus, even in the dividing cells (Figure 7).