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

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.

Genetic variation of Chinese PRRSV strains based on ORF5 sequence.

Thirteen isolates of porcine reproductive and respiratory syndrome virus (PRRSV) from different provinces of China were studied and compared with several PRRSV isolates from other countries. Phylogenetic analysis shows that all Chinese isolates of PRRSV in this study belong to the American genotype, except for one strain, B13, which clustered as a European genotype. Sequence analysis revealed that PRRSV Chinese isolates of the American genotype were highly similar in the ORF5 sequence and could be classified into two subclades. One contains PRRSV isolates that are more closely related to the American vaccine strain MLV Resp and its parent strain VR-2332, and the other contains ones only distantly related to them. Within the Chinese isolates slight genetic variation occurred, and some strains may originate directly from the vaccine virus.

Computational prediction of microRNA genes in silkworm genome.

MicroRNAs (miRNAs) constitute a novel, extensive class of small RNAs (approximately 21 nucleotides), and play important gene-regulation roles during growth and development in various organisms. Here we conducted a homology search to identify homologs of previously validated miRNAs from silkworm genome. We identified 24 potential miRNA genes, and gave each of them a name according to the common criteria. Interestingly, we found that a great number of newly identified miRNAs were conserved in silkworm and Drosophila, and family alignment revealed that miRNA families might possess single nucleotide polymorphisms. miRNA gene clusters and possible functions of complement miRNA pairs are discussed.

Expression of cholera toxin B subunit and assembly as functional oligomers in silkworm.

The nontoxic B subunit of cholera toxin (CTB) can significantly increase the ability of proteins to induce immunological tolerance after oral administration, when it was conjugated to various proteins. Recombinant CTB offers great potential for treatment of autoimmune disease. Here we firstly investigated the feasibility of silkworm baculovirus expression vector system for the cost-effective production of CTB under the control of a strong polyhedrin promoter. Higher expression was achieved via introducing the partial non-coding and coding sequences (ATAAAT and ATGCCGAAT) of polyhedrin to the 5' end of the native CTB gene, with the maximal accumulation being approximately 54.4 mg/L of hemolymph. The silkworm bioreactor produced this protein vaccine as the glycoslated pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB. Further studies revealed that mixing with silkworm-derived CTB increases the tolerogenic potential of insulin. In the nonconjugated form, an insulin : CTB ratio of 100 : 1 was optimal for the prominent reduction in pancreatic islet inflammation. The data presented here demonstrate that the silkworm bioreactor is an ideal production and delivery system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes and CTB functions as an effective mucosal adjuvant for oral tolerance induction.

Isolation and partial characterization of a novel pollen-specific cDNA with multiple polyadenylation sites from wheat.

A novel pollen-specific full-length cDNA clone PSG076 was isolated using suppression subtractive hybridization and 5'/3' RACE techniques. PSG076 was shown to exhibit multi-site polyadenylation by sequencing the 3' ends of the cDNAs. At least six transcripts with different length were produced from the single gene based on different poly(A) tail attachment sites. However, polyadenylation consensus sequence AAUAAA was not seen at the 3'-untranslated sequence. PSG076 contained a 299 bp 5' untranslated region and an open reading frame of 663 bp encoding a 221 amino acid peptide with pI of 4.31. A blast search revealed that this sequence did not show a significant similarity to any genes deposited in the public database. Southern blot indicated that PSG076 was a single copy gene. Northern blot and RT-PCR analysis indicated that PSG076 transcripts showed specific expression in mature pollen, and weak or undetectable signals in uninucleate microspore, immature seed, stem, young leave, root and ovary. Further analysis of the expression pattern in gametophyte showed that PSG076 transcripts were undetectable in uninucleate, binucleate microspore and pollen at early stage, and were first detectable and increased rapidly at middle and late stages of pollen development with the maximum level in mature pollen and also expressed in germinating pollen in vivo, suggesting that PSG076 might play a role in pollen germination and pollen tube growth in addition to its function in maturation. The evidences gathered in this work indicated that the six different transcripts from the single gene were differentially expressed during pollen development.

Expression and identification of mutated osteoprotegerin in culture cells and larvae of silkworm.

The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.

A novel tri-primer PCR method (TP-PCR) for rapid construction of fpg gene.

A novel tri-primer polymerase chain reaction method (TP-PCR) was developed for the construction of a fused fpg gene, in which no endonuclease and ligase were used. Instead, two templates and three specifically designed primers were applied. Results showed that pheB and gfp genes, which encodes the catechol 2, 3-dioxygenase and the green fluorescent protein (GFP), respectively, were successfully fused into an fpg gene through the rapid TP-PCR system, indicating that TP-PCR method could be a useful tool for DNA fragment fusion in which no proper endonuclease sites were available.

[Construction of a fused fpg gene by using TP-PCR method].

TP-PCR,a method developed for fusion gene construction without the use of endonuclease and ligase, was performed to construct a fused fpg gene. The TP-PCR reaction system contained three primers and two templates and resulting PCR product, fused fpg gene, consisted of three sections: pheB gene, which was responsible for catechol 2,3-dioxygenase, gfp gene for GFP protein and the intermediate ligation segment which was designed for the correct expression of the fusion gene. The result in this paper showed that the TP-PCR method is one of rapid and convenient methods for fused gene construction.

[Expression of polyprotein of infectious bursal disease virus in Bombyx mori].

Segment A of the genome of infectious bursal disease virus(IBDV) encodes structure protein VP2 and VP3 and protease VP4. In this study a polyprotein gene of IBDV was inserted into a Bombyx mori baculovirus transfer vector pAcHLT--C and contransfected into BmN cells with linear genome DNA of virus Bm-BacPAK6. Dot hybridization suggested that the segment A of the virus genome was inserted in the genome of Bm-BacPAK6. The silkworm of fifth instars were infected by the recombinant virus and the immunogenicity of the infected larvae's blood were examined with ELISA, SDS-PAGE and Western blotting. It appears that the recombinant polyprotein has the property of immunoreactivity and the expression in larvae reached the pick 5-day post infection.

Expression and activity determination of human angiostatin (k1-3) in culture cells and larvae of silkworm.

Angiostatin (k1-3) gene was inserted into Bombyx mori baculovirus transfer vector pBacPAK8 and cotransfected with lineared DNA of Bm-BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus BacPAK-angiostatin was expressed, as identified by DNA dot blotting. The BmN cells and fifth instars were infected by the recombinant virus BacPAK-angiostatin; expression product was run in the SDS-PAGE, and its immunoreactivity was determined by using ELISA and Western blotting. The bio-activity of the protein product was determined by using human umbilical vein endothelial cells ( ECV304 ) proliferation test in vitro and by using CAM vascular inhibition test in vivo. The expression activity achieved the highest point at the 72nd hour in BmN cells (22 u / 2x10(6) cells) and at 144th hour in larvae (159 u/ml). At the concentration of 2.5 u/ml, angiostatin induced apoptosis of endothelial cells in 24 h and also inhibited angiogenesis in CAM.

[Expression of human interleukin-11 in cell culture and larvae of silkworm].

A recombinant transfer vector, pBacIL-11, containing hIL-11 cDNA of 546 nucleotides lacking leader sequence was constructed and co-transfected into BmN cells with linearized BmBacPAK(modified BmNPV) DNA for construction of a recombinant baculovirus carrying the hIL-11 gene. Southern hybridization analysis suggested that the recombinant baculovirus DNA contained hIL-11 cDNA fragment. RNA dot blotting demonstrated that the hIL-11 gene was transcribed. The recombinant baculovirus has a strong infectivity to BmN cell line and to silkworm larvae and pupae. Specific hIL-11 bands were detected from all the samples of cell extract, culture supernatant, haemolymph of larvae and pupae by SDS-PAGE analysis. Biological activity of the expressed product was determined with IL-11 dependent B9-11 cell line and by MTT colorimetric assay, which indicated that biologically active rhIL-11 protein was overexpressed in BmN cell line and in silkworm larvae and pupae.

Infectious Bursal Disease Virus Structural Protein VP2 Expressed by a Baculovirus Recombinant in Bombyx mori.

VP2 cDNA gene of the infectious bursal disease virus HZ96 strain, encoding a major host-protective antigen, was cloned into baculovirus transfer vector pBacPAK8, resulting in a recombinant transfer vector pBacPAK-VP2. The vector pBacPAK-VP2 and linearized DNA of modified baculovirus Bm-BacPAK6 were co-transfected into the cultured Bombyx mori (Bm) N cells, in which homologous recombination occurred. Then, baculovirus recombinants were screened out. The Bm cells and Bm larvae were infected with the baculovirus recombinant that can expresse VP2, and Bm N cells and haemolymph of Bm larvae were collected for assays. The results of ELISA and Western immunoblotting assays demonstrated that VP2 was expressed in the cultured Bm cells and the Bm larvae.