Hnf4α is a key gene that can generate columnar metaplasia in oesophageal epithelium.

Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.

Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity of DNA vaccines depends, to a large extent, on the presence of CpG motifs as built-in adjuvants, we addressed this issue by inserting three types of human CpG motifs (A-type, B-type, and C-type) into the backbone of nonviral DNA and viral DNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects of CpG modifications in DNA vaccines expressing three types of antigens (ß-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types of CpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotype A and Bacillus anthracis A16R challenge. Taken together, these results demonstrate that insertion of human CpG motifs can differentially modulate the immunogenicity of nonviral DNA vaccines as well as viral DNA replicon vaccines. Our study provides not only a better understanding of the in vivo activities of CpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants for DNA vectors targeting specific antigens.

Developmental stalling and organ-autonomous regulation of morphogenesis.

Timing of organ development during embryogenesis is coordinated such that at birth, organ and fetal size and maturity are appropriately proportioned. The extent to which local developmental timers are integrated with each other and with the signaling interactions that regulate morphogenesis to achieve this end is not understood. Using the absolute requirement for a signaling pathway activity (bone morphogenetic protein, BMP) during a critical stage of tooth development, we show that suboptimal levels of BMP signaling do not lead to abnormal morphogenesis, as suggested by mutants affecting BMP signaling, but to a 24-h stalling of the intrinsic developmental clock of the tooth. During this time, BMP levels accumulate to reach critical levels whereupon tooth development restarts, accelerates to catch up with development of the rest of the embryo and completes normal morphogenesis. This suggests that individual organs can autonomously control their developmental timing to adjust their stage of development to that of other organs. We also find that although BMP signaling is critical for the bud-to-cap transition in all teeth, levels of BMP signaling are regulated differently in multicusped teeth. We identify an interaction between two homeodomain transcription factors, Barx1 and Msx1, which is responsible for setting critical levels of BMP activity in multicusped teeth and provides evidence that correlates the levels of Barx1 transcriptional activity with cuspal complexity. This study highlights the importance of absolute levels of signaling activity for development and illustrates remarkable self-regulation in organogenesis that ensures coordination of developmental processes such that timing is subordinate to developmental structure.

Binding activity and immunogenic characterization of recombinant C-terminal quarter and half of the heavy chain of botulinum neurotoxin serotype A.

In the present study, we explored and compared the binding activity and immunogenic characterization of the most effective part corresponding to C-terminal quarter of heavy chain of botulinum neurotoxin serotype A (AHc-C) with C-terminal half of heavy chain of botulinum neurotoxin serotype A (AHc). Firstly, the fully soluble AHc-C protein successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx) was shown to bind with ganglioside as the AHc, indicating that the recombinant AHc-C protein retains a functionally active conformation. Furthermore, a solid-phase assay showed that the anti-AHc-C sera effectively inhibited the binding of AHc or AHc-C to the ganglioside GT1b, the first step in BoNT/A intoxication of neurons, as good as the anti-AHc sera. Finally, although the recombinant AHc-C protein still induced a high serum antibody titers and afforded protection level as the mice challenged with active botulinum neurotoxin serotype A, the immunization with AHc protein induced stronger protective potency than the AHc-C protein. The data presented in the report shows that there are the same ganglioside binding activity and different immunogenic characterization between the C-terminal quarter and half of heavy chain of botulinum neurotoxin serotype A. Therefore, the recombinant AHc-C protein can not only be developed into a minimal subunit candidate vaccine for prophylaxis against botulinum neurotoxin serotype A but also be used as a promising tool in the search for binding inhibitors and chimeric vaccines.

Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant.

Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F.

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab')(2) fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab')(2) antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab')(2) antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.

Barrett's metaplasia: molecular mechanisms and nutritional influences.

Barrett's metaplasia is discussed in the context of a general theory for the formation of metaplasias based on developmental biology. The phenotype of a particular tissue type becomes established during embryonic development by the expression of a specific set of transcription factors. If this combination becomes altered, then the tissue type can be altered. Such events may occur by mutation or by environmental effects on gene expression, normally within the stem cell population of the tissue. A macroscopic patch of metaplastic tissue will arise only if the new gene activity state is self-sustaining in the absence of its original causes, and if the new tissue type can outgrow the parent tissue type. An important candidate gene for the causation of Barrett's metaplasia is Cdx2 (Caudal-type homeobox 2). In normal development, this is expressed in the future intestine, but not the future foregut. Mouse knockout studies have shown that it is needed for intestinal development, and that its loss from adult intestine can lead to squamous transformations. It is also expressed in Barrett's metaplasia and can be activated in oesophageal cell cultures by treatment with bile acids. We have investigated the ability of Cdx2 to bring about intestinal transformations in oesophageal epithelium. Our results show that Cdx2 can activate a programme of intestinal gene expression when overexpressed in HET-1A cells, or in fetal epithelium, but not in the adult epithelium. This suggests that Cdx2, although necessary for formation of intestinal tissue, is not sufficient to provoke Barrett's metaplasia in adult life and that overexpression of additional transcription factors is necessary. In terms of diet and nutrition, there is a known association of Barrett's metaplasia with obesity. This may work through an increased risk of gastro-oesophageal reflux. Acid and bile are known to activate Cdx2 expression in oesophageal cells. It may also increase circulating levels of TNFalpha (tumour necrosis factor alpha), which activates Cdx2. In addition, there may be effects of diet on the composition of the bile.

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate.

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.

Improved immunogenicity and protective efficacy of a replicon DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A by electric pulses and protein boosting.

Vaccination by intramuscular injection of naked DNA is very efficient in mouse model, but immunogenicity of DNA vaccines needs to be improved in human use. Thus, we wanted to determine whether suitable electric pulses-mediated DNA delivery technology and DNA prime-protein boost regimen could improve the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc in mouse model. In this study, the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc following electric pulses were dramatically improved in Balb/c mice. Also, priming of the immune response by DNA vaccination followed by a single booster with AHc protein immunogen resulted in very high levels of ELISA and neutralization antibodies and afforded more efficient protection against botulinum neurotoxin serotype A. Therefore, these methods described here potentially provide suitable strategies in developing an efficacious vaccine against Clostridium botulinum neurotoxin serotype A.

Protection with a recombinant Hc of Clostridium Botulinum neurotoxin serotype A from Escherichia coli as an effective subunit vaccine.

A recombinant Hc of Clostridium botulinum neurotoxin serotype A (AHc) was successfully expressed in Escherichia coli for use as an antigen, and the purified AHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype A. The mice, given twice or third subcutaneous vaccinations with a dosage of 1 microg AHc mixed with Freund adjuvant, were completely protected against an intraperitoneal administration of 1,000,000 50% lethal doses (LD(50)) of neurotoxin serotype A. Following the administration of AHc using alhydrogel adjuvant via the intramuscular route, a strong protective immune response was also elicited in the vaccinated mice. A dose-response was observed in protective efficacy with increasing AHc dosage and number of vaccinations. Mice that received two injections of >or= 0.2 microg and three injections of >or= 0.04 microg were completely protected when challenged with 100,000 LD(50) of neurotoxin serotype A. These results clearly suggest that the recombinant AHc highly expressed in Escherichia coli is very efficacious in protecting against challenge with active botulinum neurotoxin serotype A in mouse model and a good subunit candidate vaccine against botulinum neurotoxin serotype A for human use.

Phenotypic analysis of tumor-infiltrating lymphocytes in hepatocellular carcinoma.

BACKGROUND/AIMS: Tumor may induce local immunosuppression and make the tumor-infiltrating lymphocytes (TILs) functionally inhibited and lose the antitumor effects. Therefore, we did the phenotypic analysis of TILs in hepatocellular carcinoma (HCC) tissues. METHODOLOGY: Lymphocytes were isolated from paired HCC tissues (TILs) and the corresponding non-tumor liver tissues (non-tumor-liver infiltrating lymphocytes, NILs) of 28 patients and were subjected to flow cytometric analysis. RESULTS: Compared with the non-tumor portion, HCC tissues had less intensity of lymphocyte infiltration. TILs had higher CD3+/CD56+ ratio than NILs. Around 70%-90% NILs or TILs expressed the antigen-experienced or memory phenotypes. Around 60%-70% CD4+ or CD8+ NILs and TILs expressed the activation markers CD69 and HLA-DR. However, we found that CD25 is under-expressed in both the CD8+ NILs and TILs. In addition, more percentage of CD4+ CD25+ T cells was detected in the TILs than in the NILs. CONCLUSIONS: HCC tissues had less lymphocytes infiltration. Most of infiltrating lymphocytes from the HCC tissues and the corresponding non-tumor liver tissues were activated and expressed antigen-experienced phenotypes. However, the CD25 was underexpressed in the CD8+ TILs and the CD4+ CD25+ T cells were increased in the TILs. These factors might impair the antitumor immunity in HCCs.

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes.

BACKGROUND: Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. RESULTS: The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. CONCLUSION: We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

[In vitro molecular evolution of humanized mouse ScFv specific against human cross-linked fibrin].

In early work at our laboratory, a mouse ScFv All specific against human cross-linked fibrin was picked out by using phage displaying technique, and it was useful in prepareing targeting thrombolytic agent. To reduce its immunogenicity, All was humanized according to the surface reshaping approach. In order to increase the affinity and specifity of humanized ScFv, the Mixed HCDR3 and LCDR3 library was constructed and selected, and five different humanized ScFv with better affinity or specifity than humanized ScFv were identified, but the affinity of these five humanized scFvs was still lower than parent mouse scFv All. In this study, in order to further increase the affinity of humanized scFv, the identified five different CDR3 mutant of humanized ScFvs were mutated and reassembled with the methods of error-prone PCR and DNA shuffling, then the shuffled ScFvs were cloned into plasmid pHB-1 HSCFV to construct ScFv library of 10(5). The ScFv library was displayed on the surface of phage and panned with antigen DD, then five ScFvs with better affinity or specifity were identified by using ScFv-alkaline phosphatase detection system which was established in our laboratory. Based on identified five better ScFv, the second round error-prone PCR and DNA shuffling program was taken out, a ScFv library of 10(6) was constructed and selected as before. And four humanized ScFv that had evidently better affinity than parent mouse ScFv was identified. This work laid a foundation for further research of targeting thrombolitic agent with low immunogenicity.

Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate.

The receptor-binding domain of tetanus toxin (THc), which mediates the binding of the toxin to the nerve cells, is a candidate subunit vaccine against tetanus. In this study one synthetic gene encoding the THc was constructed and highly expressed in Escherichia coli by co-expression with thioredoxin (Trx). The purified THc-vaccinated mice were completely protected against an active toxin challenge in mouse models of disease and the potency of two doses of THc was comparable to that of three doses of toxoid vaccine. And a solid-phase assay showed that the anti-THc sera inhibited the binding of THc or toxoid to the ganglioside GT1b as the anti-tetanus toxoid sera. Furthermore, mice were vaccinated once or twice at four different dosages of THc and a dose-response was observed in both the antibody titer and protective efficacy with increasing dosage of THc and number of vaccinations. The data presented in the report showed that the recombinant THc expressed in E. coli is efficacious in protecting mice against challenge with tetanus toxin suggesting that the THc protein may be developed into a human subunit vaccine candidate designed for the prevention of tetanus.

Isolation, culture, and characterisation of mouse embryonic oesophagus and intestine.

The gastrointestinal tract of vertebrates is lined by epithelium that develops from the endodermal germ layer. The oesophagus and intestine form part of the gastrointestinal tract and studying the normal development of both tissues is difficult due to lack of suitable in vitro model systems. One of the criteria for a reliable culture model includes the ability to carry out real-time observations in vitro. The method we describe here is based on the isolation of embryonic oesophagus and intestine from 11.5-day-old embryos and culture on fibronectin-coated coverslips in basal Eagle's medium and 20% fetal bovine serum. This model permits real-time observations of both tissues and over a few days in culture, markers of differentiation appear. This culture system appears to recapitulate normal oesophagus and intestine development.

Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system.

Random integration linking genomic amplification is widely used to generate desired cell lines for stable and high-level expressing recombinant proteins. But this technique is laborious, and the expression level is unpredictable due to position effects. After reading many reports on gene amplification, we hypothesized that there should be several loci in the genome of Chinese hamster ovary (CHO) cells that allow not only high-level, but also stable gene expression. Based on this hypothesis, we constructed a plasmid pMCEscan, which introduces a site-specific recombinase-recognition sequence, FRT, for gene targeting into those sites. Another targeting vector, pcDNA5/FRT, has an FRT sequence fused to a promoterless hygromycin-resistance gene that can be expressed only when correct gene targeting occurs. Using the pMCEscan, which is a novel and stringent selection system used to create a few high protein-producing clones, we constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene targeting. We selected 28 CHO strains that expressed a high-level of reporter genes and carried one copy of the pMCEscan in their chromosomes, and we treated these strains with methotrexate (MTX) to evaluate dihydrofolate reductase (DHFR)-mediated gene amplification. Nine clones showed high-level tissue plasminogen activator (tPA) production without amplification. We then targeted other genes (tPA, secreted alkaline phosphatase (SEAP), erythropoietin (EPO)) to test the basal expression ability of nine CHO strains. CHO strains 8-1 and 8-11 consistently expressed high basal levels of the recombinant genes. Using this cell-vector system, we obtained the tPA stable high producers by gene targeting and gene amplification. This system allows for rapid generation of recombinant proteins without cloning and greatly simplifies selection of cell lines for the production of potential therapeutic proteins.

Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.

Development and preclinical evaluation of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A.

Concern about the malicious applications of botulinum neurotoxin has highlighted the need for a new generation of safe and highly potent antitoxins. In this study, we developed and evaluated the preclinical pharmacology and safety of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A (BoNT/A). As an alternative to formalin-inactivated toxoid, the recombinant Hc domain of botulinum neurotoxin serotype A (rAHc) was used to immunize horses, and the IgGs from the hyperimmune sera were digested to obtain F(ab')2 antitoxin. The protective effect of the new F(ab')2 antitoxin against BoNT/A was determined both in vitro and in vivo. The results showed that the F(ab')2 antitoxin could prevent botulism in mice challenged with BoNT/A and effectively delayed progression of paralysis from botulism in the therapeutic setting. The preclinical safety of the new F(ab')2 antitoxin was also evaluated, and it showed neither harmful effects on vital functions nor adverse effects such as acute toxicity, or immunological reactions in mice and dogs. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/A, and our findings support the safety of the new F(ab')2 antitoxin for clinical use. Our study further demonstrates the proof of concept for development of a similar strategy for obtaining potent antitoxin against other BoNT serotypes.

[Construction of mammalian cell lines continuously expressing JEV structural proteins].

Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.