Biomedical subjects

Lian Yu

Publications and source records attributed to Lian Yu.

32 records · Page 2Linked to original sources

PubMed2005

Preparation of ChIL-2 and IBDV VP2 fusion protein by baculovirus expression system.

This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2 was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.

Animals↗

PubMed2005

[Cloning, expression and preparation of polyclonal antibody for IBDV non-structure protein gene].

Infectious bursal disease virus (IBDV) VP5 gene was amplified and cloned into an N-terminal GST-Tag fusion expression vector, pGEX-4T-2, which was controlled by T7 promoter. The sequencing result showed that the VP5 gene was composed of 438 base pairs, and coded 145 amino acids. High VP5 product was expressed in E. coli BL21 induced by IPTG, and the GST-VP5 fusion protein existed in inclusion. High titer anti-VP5 serum was also prepared in New Zealand rabbit immunized with purified fusion protein inclusion. These results gave a basis for further research for VP5 function in IBDV replication and pathogenicity, which also paved the way for developing VP5 gene deleted IBDV live vaccine.

Animals↗

PubMed2004

Enhancement of the immunogenicity of DNA vaccine against infectious bursal disease virus by co-delivery with plasmid encoding chicken interleukin 2.

The immunoregulatory activity of a nonmammalian interleukin 2 (IL-2), chicken IL-2 (chIL-2), was investigated using a DNA vaccine against infectious bursal disease virus (IBDV) as a model. Coadministration of a plasmid encoding the VP2 gene of IBDV (pCI-VP2) and a plasmid encoding chicken IL-2 gene (pCI-chIL-2) enhances bursal protection against both the homologous IBDV strain ZJ2000 and the heterologous strain BC6/85 compared to administration of pCI-VP2 alone. Vaccination with pCI-VP2 alone induces low bursal protection against ZJ2000 and only protects chickens from clinical outbreaks and mortality, but not from bursal damage caused by BC6/85. Co-administration of the plasmid encoding the polyprotein gene of IBDV (pCI-VP2/4/3) and pCI-chIL-2 provides complete protection (15/15) against ZJ2000 and satisfactory protection (13/15) against BC6/85. In contrast, only 10 out of 15 chickens and 6 out of 15 chickens were protected against ZJ2000 and BC6/85, respectively, using the pCI-VP2/4/3 vaccination alone. A significant increase in the IBDV-specific neutralizing antibody response was also observed in chickens that received pCI-VP2/4/3 plus pCI-chIL-2 as compared with those that received the pCI-VP2/4/3 vaccination alone. By administrating different amounts of plasmid DNA, we confirmed that the pCI-chIL-2, but not the backbone plasmid pCI, contributes to increased immunoprotection of DNA vaccine against IBDV. These results strongly indicate that the efficacy of avian DNA vaccine can be modulated by co-administration of a plasmid encoding chIL-2.

Adjuvants, Immunologic↗

PubMed2004

[The reverse genetics systems for human and animal RNA viruses].

The recovery of the virus from genetic materials in in vitro culture systems or sensitive animals is called virus rescue. A functional infectious clone of RNA virus provides unlimited possibility for genetic studies and the related reverse genetics system that allows directed genetic manipulation of an RNA virus is an extremely powerful research tool. In the past twenty years, especially since the first infectious clone of a negative-stranded RNA virus was reported in the mid-1990's, the reverse genetics systems have been available for nearly all the major human and animal RNA virus groups. The article reviews the progress of this technology, highlighting the obstacles in the construction of reverse genetics systems for major groups of human as well as animal RNA viruses and how the virologists overcame them. There are mainly four external expression systems for construction of the RNA virus reverse genetics systems basing on the kind of RNA viruses. These systems include in vitro RNA transcripts, RNA polymerase I-driven expression plasmids, RNA polymerase II-driven expression plasmids and modified vaccinia virus/T7 RNA polymerase-driven expression system. In particular, the viral nucleoprotein and polymerase proteins are required to assemble the viral ribonucleoprotein (RNP) complexes for the rescue of the negative-stranded RNA viruses. Relevant topics about the rescue of the typical viruses are discussed, including poliovirus with the de novo synthesis, Coronaviridae with the largest size of genome, Flaviviridae with the instable clones, HCV with the quasispecies nature, nodaviruses with the virus-host interaction, influenza virus with the RNA pol I transcription system, Arenavirdae with the ambisense coding strategies etc.

Animals↗

PubMed2004

[Expression of the infectious bursal disease virus polyprotein in Vero cells using attenuated Salmonella typhimurium as transgenic carrier].

To examine if polyprotein gene (VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam- and phoP-), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.

Animals↗

PubMed2003

Nucleation of one polymorph by another.

Nucleation of one polymorph by another has been observed directly in the melt crystallization of d-mannitol and d-sorbitol. The new polymorph nucleated on an existing one and grew to dominate the end product. This effect is relevant to controlling polymorphism in the manufacture of specialty chemicals and to developing theories of polymorphic nucleation and concomitant polymorphs, for which current efforts emphasize homogeneous nucleation of alternative polymorphs.

Journal Article↗

PubMed2003

Plasmid DNA encoding antigens of infectious bursal disease viruses induce protective immune responses in chickens: factors influencing efficacy.

The complete polyprotein (VP2/4/3) and VP2 genes of two infectious bursal disease viruses (IBDVs) (one attenuated strain JD1 and one virulent strain ZJ2000) were amplified by long and accurate polymerase chain reaction (LA-PCR), cloned, sequenced and inserted into plasmids pCI and pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. A series of DNA vaccine preparations were made using liposome as the adjuvant to examine their immunogenicity. Although VP2 is the main protective immunogen of IBDV, DNA encoding VP2 initiated a very low level of neutralizing antibody and only protected chickens from clinical outbreak and morality, but not bursal damage. In contrast, DNA encoding VP2/4/3 induced neutralizing antibody and satisfactory protection against virulent IBDV. Recombinant plasmids encoding the polyprotein gene of strain ZJ2000 were more efficient at inducing an immune response than that of strain JD1. Polyprotein expressed by the pCI vector induced better immune response than that expressed by the pcDNA3. Delivery of DNA through intramuscular and/or intradermal routes elicited much higher protective responses than that of oral and eyedrop routes. Most of the chickens vaccinated with high doses of DNA were protected from challenge. Additionally, the immune response to the DNA vaccine was significantly enhanced by a liposome adjuvant. These results indicate that the source of the target genes (from different IBDV strains), the eukaryotic expression vector, the adjuvant, the delivery route and the dosage might play a role of varying degree in influencing the efficacy of the DNA vaccine against IBDV.

Animals↗

PubMed2003

Effective inhibition of mannitol crystallization in frozen solutions by sodium chloride.

PURPOSE: The purpose of this work was to study the possibility of preventing mannitol crystallization in frozen solutions by using pharmaceutically acceptable additives. METHODS: Differential scanning calorimetry (DSC) and low-temperature X-ray diffractometry (LTXRD) were used to characterize the effect of additives on mannitol crystallization. RESULTS: DSC screening revealed that salts (sodium chloride, sodium citrate, and sodium acetate) inhibited mannitol crystallization in frozen solutions more effectively than selected surfactants, alpha-cyclodextrin, polymers, and alditols. This finding prompted further studies of the crystallization in the mannitol-NaCl-water system. Isothermal DSC results indicated that mannitol crystallization in frozen solutions was significantly retarded in the presence of NaCl and that NaCl did not crystallize until mannitol crystallization completed. Low-temperature X-ray diffractometry data showed that when a 10% w/v mannitol solution without additive was cooled at 1 degrees C/min, the crystalline phases emerging after ice crystallization were those of a mannitol hydrate as well as the anhydrous polymorphs. In the presence of NaCl (5% w/v), mannitol crystallization was suppressed during both cooling and warming and occurred only after annealing and rewarming. In the latter case however, mannitol did not crystallize as the hydrate, but as the anhydrous delta polymorph. At a lower NaCl concentration of 1% w/v, the inhibitory effect of NaCl on mannitol crystallization was evident even during annealing at temperatures close to the Tg' (-40 degrees C). A preliminary lyophilization cycle with polyvinyl pyrrolidone and NaCl as additives rendered mannitol amorphous. CONCLUSION: The effectiveness of additives in inhibiting mannitol crystallization in frozen solutions follows the general order: salts > alditols > polyvinyl pyrrolidone > alpha-cyclodextrin > polysorbate 80 approximately polyethylene glycol approximately poloxamer. The judicious use of additives can retain mannitol amorphous during all the stages of the freeze-drying cycle.

Calorimetry, Differential Scanning↗

PubMed2003

Crystallization of D-mannitol in binary mixtures with NaCl: phase diagram and polymorphism.

PURPOSE: To study the crystallization, polymorphism, and phase behavior of D-mannitol in binary mixtures with NaCl to better understand their interactions in frozen aqueous solutions. METHODS: Differential scanning calorimetry, hot-stage microscopy, Raman microscopy, and variable-temperature X-ray diffractometry were used to characterize D-mannitol-NaCl mixtures. RESULTS: NaCl and D-mannitol exhibited significant melt miscibility (up to 7.5% w/w or 0.20 mole fraction of NaCl) and a eutectic phase diagram (eutectic composition 7.5% w/w NaCl; eutectic temperature 150 degrees C for the alpha and beta polymorphs of D-mannitol and 139 degrees C for the delta). The presence of NaCl did not prevent mannitol from crystallizing but, depending on sample size, affected the polymorph crystallized: below 10 mg, delta was obtained; above 100 mg, alpha was obtained. Pure mannitol crystallized under the same conditions first as the delta polymorph and then as the a polymorph, with the latter nucleating on the former. KCl showed similar eutectic points and melt miscibility with D-mannitol as NaCl. LiCl yielded lower eutectic melting points, inhibited the crystallization of D-mannitol during cooling, and enabled the observation of its glass transition. CONCLUSIONS: Despite their structural dissimilarity, significant melt miscibility exists between D-mannitol and NaCl. Their phase diagram has been determined and features polymorph-dependent eutectic points. NaCl influences the polymorphic behavior of mannitol, and the effect is linked to the crystallization of mannitol in two polymorphic stages.

Algorithms↗

PubMed2003

[Rapid construction of infectious clones of infectious bursal disease virus].

A rapid procedure was established for rescuing infectious bursal disease virus (IBDV), an important pathogen in poultry. A full-length cDNA clone of the segment B of a CEF-adapted IBDV strain HZ2 was constructed by long RT-PCR, and the 2827 bp nucleotide sequence, including the 5 - and 3 -noncoding regions (NCR), was established. Then the cDNA clone of segment B was engineered to make it contain three silent nucleotide changes, creating a new EcoRV site that was different from the parent virus sequences, by site-directed silent mutagenesis. Cotransfection of eukaryotic expression recombinants containing modified segment A and segment B with Lipofectamine into Vero cells resulted in the expression of IBDV RNA and proteins, as confirmed by Northern RNA dot hybridization and indirect immunofluorescence assay analysis. The change of cell morphology after cotransfection and passages of cell cultures was similar to that of cells infected by authentic IBDV, causing cellular pathogenic effects (CPE). The virus-like particles at 55-60 nm were observed under electron microscopy, affirming the rescue of IBDV. The genetic markers were retained in the recovered progeny virus.

Animals↗

PubMed2002

Glycine crystallization during spray drying: the pH effect on salt and polymorphic forms.

Spray drying of aqueous solutions of glycine revealed a strong pH effect on the salt and polymorphic forms of the resulting powders. Adjusting pH by aqueous HCl or NaOH between 1.7 and 10.0 caused the glycine solutions to crystallize as two polymorphs (alpha and gamma) of the neutral glycine ((+)H(3)NCH(2)CO(2) (-)) and as three salts (diglycine HCl, (+)H(3)NCH(2)CO(2) (-). (+)H(3)NCH(2)CO(2)H. C1(-); glycine HCl, (+)H(3)NCH(2)CO(2)H. C1(-); and sodium glycinate, H(2)NCH(2)CO(2) (-). Na(+)). Although alpha-glycine crystallized from solutions without pH adjustment (pH 6.2), changing the pH to 4.0 and 8.0 caused gamma-glycine to crystallize as the preferred polymorph. This phenomenon is attributed to the pH effect on the dimeric growth unit of alpha-glycine. The formation of alpha-glycine by spray drying solutions of neutral glycine contrasts the outcome of freeze drying, which yields beta-glycine. Because gamma-glycine is thermodynamically more stable than alpha-glycine, the crystallization of gamma-glycine by pH adjustment provides a way to improve the physical stability of glycine-containing formulations. Spray drying at low pH yielded various mixtures of neutral glycine and its HCl salts: pH 3.0, gamma-glycine and diglycine HCl; pH 2.0, diglycine HCl; and pH 1.7 (the natural pH of glycine HCl), diglycine HCl (major component) and glycine HCl (minor component). Spray drying glycine HCl solutions (pH 1.7) yielded the same diglycine HCl/glycine HCl mixture as did spray drying neutral glycine solutions acidified to pH 1.7. Obtaining diglycine HCl by spray drying glycine HCl solutions indicates a 50% loss of HCl during processing. The extent of HCl loss could be altered by changing the inlet temperature of the spray drier. Spray drying glycine solutions at pH 9.0 and 10.0 gave predominantly gamma-glycine and an additional crystalline product, possibly sodium glycinate. The glycine powders spray dried at different pH had different particle morphologies and sizes, which may influence their suitability for pharmaceutical formulations.

Crystallization↗

PubMed2002

[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.

Animals↗

PubMed2001

Amplification and Cloning by Long RT-PCR of Full-length Genome of Larger Segment of Chicken Infectious Bursal Disease Virus.

To develop the genetic rescue techniques for infectious bursal disease virus (IBDV), Birnaviridae family, the full-length cDNA of the larger segment of the chicken IBDV was amplified and cloned by long RT-PCR. A comparison of four purification and extraction methods of RNA from IBDV infected chicken embryoid fibroblasts (CEF) showed that the ultracentrifugation followed by proteinase Kdigestion extracted dsRNA more effectively. Then reverse transcription was carried out at 50 degrees using Superscipt II enzyme, followed by RNase H digestion. Amplification of single stranded cDNA in a single step resulted in the synthesis of the full-length segment A of 3 259 bp. The amplified product was cloned and sequenced, identifying that it was an IBDV. This method is superior to other methods based on amplifying different parts of the genome many times, therefore the cloning procedure was simplified.

Journal Article↗

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↗