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author:("Yi, yongxiu")
1.  Development of a Foot-and-Mouth Disease Virus Serotype A Empty Capsid Subunit Vaccine Using Silkworm (Bombyx mori) Pupae 
PLoS ONE  2012;7(8):e43849.
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae (Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID50). Furthermore, the 50% bovine protective dose (PD50) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD50 per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.
PMCID: PMC3428285  PMID: 22952788
2.  Transcriptome Analysis of the Silkworm (Bombyx mori) by High-Throughput RNA Sequencing 
PLoS ONE  2012;7(8):e43713.
The domestic silkworm, Bombyx mori, is a model insect with important economic value for silk production that also acts as a bioreactor for biomaterial production. The functional complexity of the silkworm transcriptome has not yet been fully elucidated, although genomic sequencing and other tools have been widely used in its study. We explored the transcriptome of silkworm at different developmental stages using high-throughput paired-end RNA sequencing. A total of about 3.3 gigabases (Gb) of sequence was obtained, representing about a 7-fold coverage of the B. mori genome. From the reads that were mapped to the genome sequence; 23,461 transcripts were obtained, 5,428 of them were novel. Of the 14,623 predicted protein-coding genes in the silkworm genome database, 11,884 of them were found to be expressed in the silkworm transcriptome, giving a coverage of 81.3%. A total of 13,195 new exons were detected, of which, 5,911 were found in the annotated genes in the Silkworm Genome Database (SilkDB). An analysis of alternative splicing in the transcriptome revealed that 3,247 genes had undergone alternative splicing. To help with the data analysis, a transcriptome database that integrates our transcriptome data with the silkworm genome data was constructed and is publicly available at To our knowledge, this is the first study to elucidate the silkworm transcriptome using high-throughput RNA sequencing technology. Our data indicate that the transcriptome of silkworm is much more complex than previously anticipated. This work provides tools and resources for the identification of new functional elements and paves the way for future functional genomics studies.
PMCID: PMC3426547  PMID: 22928022
3.  Baculovirus immediately early 1, a mediator for homologous regions enhancer function in trans 
Virology Journal  2010;7:32.
Enhancers are DNA sequences that serve as binding sites for regulatory proteins, and stimulate transcriptional activity independent of their positions and orientations with respect to the transcriptional initiation site. Previous studies considered that baculovirus homologous regions (hrs) function as enhancers in cis. In our study, a plasmid containing homologous region 3 (hr3) enhancer from Bombyx mori nucleopolyhedrovirus (BmNPV) failed to enhance transcription of promoter in other plasmid in co-transfection assays, but strong stimulation occurred when cells were infected by BmNPV.
The cotransfection results of each BmNPV genomic library plasmid, hr3 plasmid and reporter plasmid showed that there were eight library plasmids stimulated the luciferase gene expression remarkably. Sequencing these plasmids revealed that each of them contained the ie-1 gene. Transfected plasmids, containing ie-1, hr3 and various origin promoter drove reporter gene showed the function was even retained. Cotransfection of hr3 functional dissected fragment and ie-1 revealed that the 30-bp imperfect palindrome destroyed fragment can't enhance reporter gene expression even though transfected with ie-1.
IE-1 was the only early factor of BmNPV that could act as a mediator for hr enhancer function in trans and the trans-function was achieved with a broad-spectrum of promoters. The 30-bp imperfect palindrome was the elementary molecular structure by which IE-1 participated in the enhancer function in trans.
PMCID: PMC2834656  PMID: 20144239
4.  Expression of Foot-and-Mouth Disease Virus Capsid Proteins in Silkworm-Baculovirus Expression System and Its Utilization as a Subunit Vaccine 
PLoS ONE  2008;3(5):e2273.
Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines.
Methodology and Principal Findings
A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD50 (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD50 per dose.
The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.
PMCID: PMC2386233  PMID: 18509464
5.  Analysis of four achaete-scute homologs in Bombyx mori reveals new viewpoints of the evolution and functions of this gene family 
BMC Genetics  2008;9:24.
achaete-scute complexe (AS-C) has been widely studied at genetic, developmental and evolutional levels. Genes of this family encode proteins containing a highly conserved bHLH domain, which take part in the regulation of the development of central nervous system and peripheral nervous system. Many AS-C homologs have been isolated from various vertebrates and invertebrates. Also, AS-C genes are duplicated during the evolution of Diptera. Functions besides neural development controlling have also been found in Drosophila AS-C genes.
We cloned four achaete-scute homologs (ASH) from the lepidopteran model organism Bombyx mori, including three proneural genes and one neural precursor gene. Proteins encoded by them contained the characteristic bHLH domain and the three proneural ones were also found to have the C-terminal conserved motif. These genes regulated promoter activity through the Class A E-boxes in vitro. Though both Bm-ASH and Drosophila AS-C have four members, they are not in one by one corresponding relationships. Results of RT-PCR and real-time PCR showed that Bm-ASH genes were expressed in different larval tissues, and had well-regulated expressional profiles during the development of embryo and wing/wing disc.
There are four achaete-scute homologs in Bombyx mori, the second insect having four AS-C genes so far, and these genes have multiple functions in silkworm life cycle. AS-C gene duplication in insects occurs after or parallel to, but not before the taxonomic order formation during evolution.
PMCID: PMC2315653  PMID: 18321391
6.  Cetyltriethylammonium bromide stimulating transcription of Bombyx mori nucleopolyhedrovirus gp64 gene promoter mediated by viral factors 
Cytotechnology  2003;41(1):37-44.
To characterize the effects of cetyltriethylammonium bromide (CTAB) on the transcription of gp64 promoter from Bombyx mori nucleopolyhedrovirus (BmNPV), the plasmid pBmgp64Luc used in transient expression assay system was constructed by using the luciferase gene as a reporter under the control of BmNPV gp64 promoter. When the Bombyx mori cells (Bm-N) were transfected with the pBmgp64Luc, different treatments were undertaken. We found that the transient expression activity of luciferase could not be augmented directly by CTAB treatment alone, but could be enhanced more than 2 times by BmNPV treatment alone at a multiplicity of infection (MOI) of 0.5. Through co-treatment with 0.1 µg ml−1 of CTAB and BmNPV at a MOI of 0.5, the enzymatic activity increased 5.21 times. We presumed that the stimulation of transcription of BmNPV gp64 promoter by CTAB was mediated by viral factors from BmNPV. In addition, the time curves of luciferase activity in cells transfected with pBmgp64Luc and transactivated by virus were observed.
PMCID: PMC3449759  PMID: 19002960
Bombyx mori nucleopolyhedrovirus; Cetyltriethylammonium bromide; gp64 promoter; Transactivition; Transfection; Transient expression

Results 1-6 (6)