Biomedical subjects

Yao-Zhou Zhang

Publications and source records attributed to Yao-Zhou Zhang.

16 recordsLinked to original sources

PubMed2006

Expression, purification and characterization of a three-domain Kazal-type inhibitor from silkworm pupae (Bombyx mori).

Serine protease inhibitors are essential for host physiological and immunological activities in insects. Analyzing the amino-acid sequence of a cDNA coding for a serine protease inhibitor in Bombyx mori (BmSPI), we found that BmSPI contained three homologous domains with a conserved sequence of C-X(3)-C-X(9)-C-X(6)-Y-X(7)-C-X(3)-C-X(11)-C similar to that of Kazal-type serine protease inhibitors, suggesting BmSPI as a new member of the Kazal-type serine protease inhibitor family. To characterize the three-domain Kazal-type inhibitor from silkworm pupae, the recombinant protein was expressed in Escherichia coli BL21 (DE3) Star. After purification with affinity and reversed-phase chromatographies, the recombinant BmSPI with a molecular mass of 33.642 Da was shown to be a specific subtilisin A inhibitor. Further studies indicated that the K(i) value of the recombinant BmSPI was 3.35 nM and the inhibitor seemed to form a 1:1 complex with subtilisin A. This is a first description of the structure and characterization of Kazal-type inhibitor with three domains cloned from silkworm pupae, B. mori.

Amino Acid Sequence↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori.

BACKGROUND: The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. RESULTS: Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. CONCLUSION: A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

Amino Acid Sequence↗

PubMed2006

The scaleless wings mutant in Bombyx mori is associated with a lack of scale precursor cell differentiation followed by excessive apoptosis.

The scaleless wings mutant in Bombyx mori (scaleless, sl) was previously reported morphologically. In the present study, we give data to clarify the mechanism of the mutation at the developmental level. Programmed cell death participates in the wing scale development during early pupal stage, and there are significant differences between that of sl and the wild type (WT) at each phase. Well-differentiated scale precursor cells do not form in sl when they have formed in WT. The peak of Caspase-3/7 activity in sl occurs 1 day later than and ten times as much as that in WT. Apoptotic bodies and DNA ladder studies also show that there is excessive apoptosis in sl early pupal wing. In addition, we have studied Bm-ASH1, an achaete-scute homolog in B.mori, which is thought to play a key role during the development of wing scales, and have found that the gene structure and expression levels of Bm-ASH1 in sl and WT are identical. All the data indicate that the wing scale precursor differentiation mechanism is abnormal in sl, which causes failing determination of scale cells and the downstream symptom of excessive apoptosis. But some of the elements to the scale differentiation circuit, such as Bm-ASH1, still operate in sl.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

[Progresses in the structure and function of Kazal-type proteinase inhibitors].

Proteinase inhibitors are widely distributed in many living organisms and play crucial roles in many biological processes, particularly in regulating the proteinase activity spatially and temporally. However, The Kazal family of serine protease inhibitors is one of the most important and extensively studied protease inhibitor families. This type of protease inhibitor normally consists of one or several domains. Every domain has a highly conserved sequence structure and molecular conformation. It is found that contact residues are hyper variable, which are responsible for the interaction of inhibitors and proteinases. Most of them are in the solvent exposed loop. But P1 residue is the key active site of the interaction between inhibitor and enzyme. The types of the amino acid at P1 site likely play an important role in causing different inhibitory activity. The substitutions at the contact residues cause significant effects on the association constant. By using the Laskowski algorithm, the Ki values of a Kazal domain against six serine proteinases can be predicted from the domain' s sequence alone. At present there are many Kazal proteinase inhibitors found in the organisms, which show important biological functions. This article gives a comprehensive review of the newer developments in the characters and the interaction of the Kazal-type inhibitors.

Amino Acid Sequence↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

[Expression and activity determination of TNFR domain of osteoprotegerin in E.coli and corresponding antibody preparation].

Osteoprotegerin (OPG) plays an important role in the regulation of bone resorption and remodeling. The TNFR domain of OPG, which is involved in the inhibition of formation and activity of osteoclasts, was amplified by PCR and inserted into multiple cloning site of PET-28a. The recombinant plasmid was transferred into E.coli BL21 to express recombinant protein. It was found that expressed product existed in the form of inclusion body. The inclusion body was solubilized, renatured and purified by affinity chromatography. Polyclonal antibodies with high specificity were obtained from the serum of rabbit immunized with purified recombinant protein. Mice were used to determine the hypocalcemic effect of the recombinant protein. Results showed that the recombinant protein expressed in E.coli had the proper bioactivity.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2003

[An in vitro nuclear run-on assay for nuclear function of angiogenin].

A sensitive and quantitative in vitro analysis method was established to detect the nascent RNAs stimulated by angiogenin using the nuclei isolated from human umbilical vein endothelial cells (HUVE). Angiogenin was mixed with nuclei in the reaction buffer, then transcription was initiated by adding the NTPs mixture. The RNA products were measured quantitatively by [alpha-(32)P]CTP incorporation with a liquid scintillation counter, either after removing free isotope by using spin column, or cutting the electrophoresis lane and counting after autoradiography. It was found that the optimum reacting temperature was 30 degrees, the most suitable reaction time was 30 min for this system, and the transcription enhancement activity of angiogenin was dose-dependent with the feasible concentration being 1 mg/L. Higher concentration of angiogenin degraded the RNA products in the system, suggesting that there is a mechanism to control the entry and accumulation of angiogenin in the target cells, which ensured angiogenin to play its role properly in the cells. Based on the evidence that angiogenin bound to DNA in nucleolus and enhanced RNA transcription, it was proposed that angiogenin might act as a trans-acting factor in nucleus to regulate RNA transcription, and inhibition of angiogenin-stimulated RNA transcription might be a promising target for screening anti-angiogenesis inhibitor.

Cell Line↗

PubMed2002

[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.

Animals↗

PubMed2002

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.

Allantois↗

PubMed2000

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.

Journal Article↗