[Construction and expression of recombinant baculovirus with Schistosoma japonicum SjPP gene].

OBJECTIVE: To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells. METHODS: The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid. The recombinant donor pFastBac-SjPP was transformed into E.coli DH10Bac forming Bacmid-SjPP which was transfected into insect cell with cational lipofectin. The fusion protein SjPP was analyzed with SDS-PAGE and Western blotting. RESULTS: The infective recombinant baculovirus Bacmid-SjPP was obtained and SjPP protein was expressed in insect cells. CONCLUSION: The recombinant protein SjPP has been expressed in insect TN5B1-4 cells with proper antigenicity.

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.

Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus.

BACKGROUND: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome. RESULTS: The genome of AnpeNPV, which infects Chinese tussah silkworm (Antheraea pernyi), was sequenced and analyzed. The genome was 126,629 bp in size. The G+C content of the genome, 53.4%, was higher than that of most of the sequenced baculoviruses. 147 open reading frames (ORFs) that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. Of the 147 ORFs, 143 appeared to be homologous to other baculovirus genes, and 4 were unique to AnpeNPV. Furthermore, there are still 29 and 33 conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. In addition, the total number of genes common to all lepidopteran NPVs is sill 74, however the 74 genes are somewhat different from the 74 genes identified before because of some new sequenced NPVs. Only 6 genes were found exclusively in all lepidopteran NPVs and 12 genes were found exclusively in all Group I NPVs. AnpeNPV encodes v-trex(Anpe115, a 3' to 5' repair exonuclease), which was observed only in CfMNPV and CfDEFNPV in Group I NPVs. This gene potentially originated by horizontal gene transfer from an ancestral host. In addition, AnpeNPV encodes two conotoxin-like gene homologues (ctls), ctl1 and ctl2, which were observed only in HycuNPV, OpMNPV and LdMNPV. Unlike other baculoviruses, only 3 typical homologous regions (hrs) were identified containing 2~9 repeats of a 30 bp-long palindromic core. However, 24 perfect or imperfect direct repeats (drs) with a high degree of AT content were found within the intergenic spacer regions that may function as non-hr, ori-like regions found in GrleGV, CpGV and AdorGV. 9 drs were also found in intragenic spacer regions of AnpeNPV. CONCLUSION: AnpeNPV belongs to Group I NPVs and is most similar to HycuNPV, EppoNPV, OpMNPV and CfMNPV based on gene content, genome arrangement, and amino acid identity. In addition, analysis of genes that flank hrs supported the argument that these regions are involved in the transfer of sequences between the virus and host.

Expression of open reading frames in silkworm pupal cDNA library.

A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open reading frame (ORF) sequences in Escherichia coli. cDNA sequences were compared to the silkworm (B. mori) genome in the GenBank database and the silkworm cDNA database including the SilkBase and KAIKOBLAST databases and 498 novel expressed sequence tags (ESTs) and 217 unknown ESTs were found. After comparison with all available ORF-complete mRNA sequences from the same organism (fruitfly, mosquito, and apis) in the RefSeq collection, 1659 full-length cDNA were identified. In addition, the structure of silkworm mRNA was analyzed, and it was found that 66.8% of silkworm mRNA tailed with poly(A) contained the highly conserved AAUAAA signal and the signal located 10-17 nucleotides upstream of the putative poly(A). Finally, the composition of nucleotides in promoter region for all ESTs was surveyed. The results imply that the TTTTA box may possess some functions in regulating transcription and expression of some genes.

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori).

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.

Oral administration activity determination of recombinant osteoprotegerin from silkworm larvae.

Osteoprotegerin (OPG) regulates the formation of osteoclasts and is involved in the regulation of bone resorption and remodeling. To investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant osteoprotegerin as a oral administration drug, the rh-OPG was expressed in the larvae of silkworm through the silkworm baculovirus expression system, and was orally administered to mice. Compared with the control, oral administration of rh-OPG was effective to decrease serum calcium concentration in normal mice, and block the bone loss induced by the loss of estrogen in ovariectomized mice. These results indicated that oral administration of rh-OPG expressed in silkworm larvae had the proper bioactivity.

Recombinant functional human lactoferrin expressed in baculovirus system.

Human lactoferrin (hLf) is a multifunctional iron-binding glycoprotein. In this study, we amplified hLf cDNA by reverse transcription-polymerase chain reaction from normal human mammary gland. The nucleotide sequence of the hLf was identical to the known hLf. We constructed a recombinant virus, vBm-hLf, harboring the hLf gene and exploited the BmN cells as host to produce recombinant human lactoferrin (rhLf). It was found that a recombinant protein with a molecular mass of approximately 78 kDa was expressed. Approximately 13.5 microg rhLf was purified from 1-2x10(5) BmN cells infected by vBm-hLf and the rhLf proved to be biologically active. This method established in our study will pave the way for efficient production of rhLf for further application of this protein in the future.

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor.

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.

The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex.

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.

High level expression of functionally active human lactoferrin in silkworm larvae.

In this paper, recombinant human lactoferrin (rhLf) was expressed very well using Bombyx mori nuclear polyhedrosis baculovirus expression system. Infection of silkworm larvae with recombinant virus, vBm-hLf, the rhLf was efficiently secreted into larvae hemolymph and the concentration of product purified was about 65 microg/ml. The isolated rhLf molecular mass was approximately 78 kDa, lower than that of the human lactoferrin (hLf) standards, which may be due to incomplete glycosylation or protein degradation. Furthermore, the rhLf was characterized and its biological activities were evaluated by in vivo bioassay using dextran sodium sulfate (DSS)-induced colitis mouse model that mimics some characteristics of colitis disease in human. We conclude that silkworm expression system can be used successfully to express functional human lactoferrin.

Cloning and identification of methionine synthase gene from Pichia pastoris.

Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.

Expression of an antimicrobial peptide identified in the male reproductive system of rats.

Bin1b is a beta-defensins-like molecule originally isolated from the rat epididymis. Owing to its bactericidal activity, Bin1b may have therapeutic properties suitable for the treatment of sexually transmitted diseases. The amino terminus of the mature Bin1b peptide contains a conserved myristoylated Gly residue. We studied the requirement of the terminal myristoylated Gly residue in the bactericidal activity of Bin1b and found that the terminal myristoylated Gly residue is not essential for the bactericidal activity. In addition, we expressed the tandem repeats of Bin1b in Escherichia coli and found that two tandem repeats of Bin1b protein were successfully expressed. The bacterially expressed tandem Bin1b repeats may be used in a diverse array of biochemical and cell biological studies.

Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus expression system. Recombinant hexa-histidine-tagged N protein with a molecular mass of 47 kD was produced in insect cells. Recombinant N protein was purified to near homogeneity by Ni2+-NTA affinity chromatography. In addition, we examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn 5B1-4 cells infected with recombinant baculovirus. The N protein was found localized in the cytoplasm as well as in the nucleolus. The purified recombinant N protein can be used in further functional study of SARS-CoV.

Production of recombinant human osteoprotegrin from Trichoplusia ni cells and Bombyx mori larvae.

Human osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant OPG-Fc were constructed and successfully expressed in Trichoplusia ni cells and Bombyx mori larvae. Native SDS-PAGE and Western blot analysis revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori larvae compared with OPG and OPG-280. Furthermore, the hypocalcemic effect assay showed that truncation of the C-terminal 100 residues OPG does not abolish the biological activity and Fc can be helpful in forming the OPG homodimer with improved biological activity.

[Research on TALF expression in Escherichia coli].

The expression of cDNA encoding Tachyleus auti-lipoposaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gram-negative bacterial endotoxin. First, the TALF gene was inserted into expression vectors pGEX-4T-2, pET22b and pET28a to construct recombinant expression plasmids. The recombinant plasmids were transformed to E. coli BL21 (DE3) and the expression of TALF was examined. Results show that TALF in pET22b and pET28a vectors can't be expressed. Only the fusion protein GST-TALF was expressed in E. coli BL21 existing as inclusion bodies. From 1 liter of culture, about 4mg of fusion protein GST-TALF with 91% purity was finally obtained. No apparent bactericidal activity and LPS neutralizing activity of the fusion protein GST-TALF were found. After digested with thrombin, the fusion protein GST-TALF exhibited strong bactericidal activity and LPS neutralizing activity.

[Expression of a human ribonuclease inhibitor variant in Escherichia coli and silkworm insect cell (Bombyx mori)].

Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR). Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence. Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori). The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system.

[Experimental research on spine fusion induced by tissue engineered bone].

OBJECTIVE: To construct a reasonable substitute for the autograft bone in vitro and transplant it back into the rabbit models to induce the spine fusion. METHODS: The bone marrow stem cell from the seven New Zealand rabbits were cultured. Recombinant human bone morphogenetic protein-4 (rhBMP-4) that has been proved to be bioactive was obtained by the way of genetic engineering. Using the vacuum freezing machine to mix a certain quantity of rhBMP-4 into type I collagen to form a new kind of carrier. Animal model of spine facet process fusion was used. Bone marrow stem cells combined with rhBMP-4 and I type collagen were implanted between the facet process to induce the spine fusion. type I collagen and bone marrow stem cell was used in the controlled group. RESULTS: New bone formation was obvious in the test group. The facet joint was fused very well in this side. No bone formation was present on the other side. CONCLUSIONS: The new composite: bone marrow stem cells, rhBMP-4 and type I collagen was an ideal kind of substitute for the autograft bone.

Human mitochondrial leucyl-tRNA synthetase with high activity produced from Escherichia coli.

The processing of human mitochondrial leucyl-tRNA synthetase had been previously investigated in insect cell. In the present work, the gene encoding human mitochondrial leucyl-tRNA synthetase with the same N-terminus as that processed in the mitochondria of insect cell was cloned and expressed in Escherichia coli. The enzyme was purified by affinity chromatography on Ni-NTA column. About 6 mg of human mitochondrial leucyl-tRNA synthetase was obtained from 1 liter of culture. The specific activity of the purified enzyme is 127.7 units/mg, the highest activity of the reported results; this enzyme has the potential for characterizing the mitochondrial tRNA mutants associated with some human mitochondrion-related neuromuscular disorders. The kinetic constants for three substrates: leucine, ATP, and E. coli tRNA1Leu (CAG) in the leucylation reaction are also reported herein.

cDNA cloning and characterization of Agkistin, a new metalloproteinase from Agkistrodon halys.

Agkistin was a new snake venom metalloproteinase (SVMP) gene which was cloned from Agkistrodon halys. Its deduced amino acid sequence has two additional cysteines (Cys407 and Cys426) in the disintegrin domain compared to other RGD containing SVMPs. The full-length gene (Agkistin) and its disintegrin region (named Agkistin-s) were expressed by baculovirus expression system (pFastBac-Htb vector) with His-tag, and their platelet aggregation-inhibition activity was evaluated. The expressed protein Agkistin can also induce apoptosis of HMEC cells in the basal medium after incubated at 37 degrees C for 20 h.

Promoter activities in the baculovirus envelope glycoprotein gp64 gene.

Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 - 22 times in Bm-N cells and over 7000 - 14,000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 - 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.