Efficient Production of Enterovirus 71 (EV71) Virus-like Particles by Controlling Promoter Strength in Insect Cells.

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

JEV-nanobarcode and colorimetric reverse transcription loop-mediated isothermal amplification (cRT-LAMP).

Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of  rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.

Influenza Virus-like Particle (VLP) Vaccines Expressing the SARS-CoV-2 S Glycoprotein, S1, or S2 Domains.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic had brought disastrous consequences throughout the entire world. While several manufactured vaccines have been approved for emergency use, continuous efforts to generate novel vaccines are needed. In this study, we developed SARS-CoV-2 virus-like particles (VLPs) containing the full length of spike (S) glycoprotein (S full), S1, or S2 together with the influenza matrix protein 1 (M1) as a core protein. Successfully constructed VLPs expressing the S full, S1, and S2 via Sf9 cell transfections were confirmed and characterized by Western blot and transmission electron microscopy (TEM). VLP immunization in mice induced higher levels of spike protein-specific IgG and its subclasses compared to naïve control, with IgG2a being the most predominant subclass. S full and S1 immune sera elicited virus-neutralizing activities, but these were not strong enough to fully inhibit receptor-ligand binding of the SARS-CoV-2. Neutralizing activities were not observed from the S2 VLP immune sera. Overall, our findings revealed that S full or S1 containing VLPs can be developed into effective vaccines.

SpyCatcher-SpyTagged ApxIA Toxoid and the Immune-Modulating Yeast Ghost Shells.

Actinobacillus pleuropneumoniaesecretes the hemolytic cytotoxins ApxI, ApxII, ApxIII, and ApxIV, which cause pleurop- neumonia in swine. Of these, ApxI is the most toxic. ApxIA, a repeats-in-toxin toxin-like protein, has strong hemolytic and cytotoxic activities. This study aimed to develop an immune modulator ApxIA toxoid, with a Spytag/Spycatcher pair (SC::ST pair), in yeast ghost shells (YGSs). These YGSs were utilized as ApxIA toxoid delivery platforms for -glucan components that can be recognized by the innate immune system. The SC::ST pair was used to conjugate the ApxIA toxoid to YGSs. The YGS-SC::ST-ToxApxIA was successfully phagocytosed by RAW 264.7 macrophages cells, without any toxicity. Further investigation revealed that YGS-SC::ST-ToxApxIA led to defective immune responses, in addition to increased levels of cytokines IL-1ß, TNF-α, and IL-10. A membrane proteomic analysis, to determine preferential major histocompatibility complex binding of ApxIA-derived peptides, was performed and four ApxIA peptides were successfully identified by liquid chromatography with tandem mass spectrometry analysis. The identified peptides may serve as poten- tial vaccine candidates in immunobiology studies of A. pleuropneumoniae. Our results indicate that YGS-SC::ST-ToxApxIA can prevent A. pleuropneumoniae pleuropneumonia (APP) by inducing both humoral and cellular immune responses.

Baculovirus entire ORF1629 is not essential for viral replication.

It is generally accepted that ORF1629 is essential for baculovirus replication, which has enabled isolation of recombinant viruses in a baculovirus expression system using linearized viral DNA. ORF1629-defective viruses cannot replicate in insect cells; only recombinant virus with complete ORF1629 restoration by recombination can propagate, allowing for pure isolation and the development of bacmids for easy selection of recombinant viruses. We inadvertently found proliferation in insect cells of a bacmid lacking a complete ORF1629. PCR indicated no other viruses but a lack of complete ORF1629 in the proliferated bacmid, suggesting that the baculovirus propagated without a complete ORF1629. Lack of ORF1629 decreased the virus growth rate and yield; it also increased the occlusion body (OB) size but decreased its yield. These results were confirmed for Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV). Thus, entire ORF1629 is not essential for viral replication, though it does affect the virus growth rate, yield, and size and OB production.

Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System.

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins.

The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions.

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.

Recent vaccine technology in industrial animals.

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.

Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter.

Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene-encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen's oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codon-optimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 5'-UTR. Subsequently, a 3'-UTR carrying the poly-A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW-derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg-derived EGF afforded slightly higher levels of re-epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct-specific minisynthetic promoter.

Production of egg yolk antibodies specific to house dust mite proteins.

PURPOSE: House dust mites (HDMs) are an important source of indoor allergens associated with asthma, rhinitis and atopic dermatitis. Chicken immunoglobulin (Ig) Y is known to be a good alternative to mice and rabbit antibody production. In this study, we produced IgYs specific to HDMs and investigated their IgE immunoreactivities. MATERIALS AND METHODS: Total IgYs were isolated from the yolks of White Leghorn hens immunized with either Dermatophagoides pteronyssinus or D. farinae protein extract. Control antibodies were separated from the yolks of immunized hens with phosphate buffered saline. IgYs specific to HDMs were analyzed using enzyme-linked immunosorbent assay and Western blotting analysis. RESULTS: The concentration of egg IgY specific to D. farinae in an immunized hen increased and the highest achieved was 661.3 ug/mg (per an egg) on day 47, compared with 760 ug/mg IgY specific to D. pteronyssinus on day 16. The D. pteronyssinus or D. farinae-specific IgY was detected by binding of each mite proteins, and their immunoreactivities were elevated dependent of the specific IgY concentration. CONCLUSION: IgY specific to HDMs may be a promising antibody for immunological diagnosis as well as identification of possible resistance relating to HDM allergy.

Regulation of glucose phosphate isomerase by the 3'UTR-specific miRNAs miR-302b and miR-17-5p in chicken primordial germ cells.

Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate in glucose pathways. Because glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In the present study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3'-untranslated region (3'UTR)-targeting miRNAs, including the gga-miR-302 cluster, gga-miR-106, gga-miR-17-5p, and gga-miR-20 cluster in chicken primordial germ cells (PGCs), compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d, and gga-miR-17-5p were expressed at lower levels than GPI mRNA. The remaining four miRNAs-gga-miR-302c, gga-miR-106, gga-miR-20a, and gga-miR-20b-were expressed at higher levels than the expression of GPI mRNA. Next, we cotransfected four candidate miRNAs-gga-miR-302b, gga-miR-106, gga-miR-17-5p, and gga-miR-20a-with GPI 3'UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p miRNAs in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGC proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affected PGCs proliferation. However, regulation of GPI using these two miRNAs did not affect the migration of PGCs into embryonic gonads.

Characterization and application of oviductal epithelial cells in vitro in Gallus domesticus.

Chicken oviductal epithelium produces large quantities of egg white protein in daily cycles. In this study, we cultured and characterized oviductal epithelial cells (OECs) from juvenile (10-wk-old) chickens and from actively laying (30-wk-old) hens. The juvenile OECs were maintained over passage 25 and were positive for toluidine blue, lectin-ConA, HPA, UEA-1, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR, whereas the adult OECs were cultured over passage 6 and were positive for toluidine blue, periodic acid-Schiff, lectin-ConA, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR. To investigate the optimal concentration of steroid hormones for inducing egg white protein genes in vitro, we examined the effects of estrogen, diethylstilbestrol, progesterone, and corticosterone on OECs. Results showed that oviduct-specific levels of avidin, ovalbumin, ovomucin, lysozyme, ESR1, and PGR gene expression were significantly elevated in steroid hormone-treated OECs compared with those of untreated cells (P < 0.05). Ovalbumin protein was also secreted into culture medium from hormone-treated OECs. In addition, to examine the application of OECs for avian transgenesis, we introduced human thrombopoietin (THPO)-expressing lentiviral vector controlled by a 3.5-kb ovalbumin promoter into cultured OECs, and THPO expression was significantly induced with diethylstilbestrol or progesterone in juvenile OECs (P < 0.05) and in adult OECs (P < 0.05). In conclusion, these data demonstrate the potential of cultured OECs as a model system for providing a better understanding of the regulation of gene expression and for the production of an avian transgenic bioreactor.

Structural and histological characterization of oviductal magnum and lectin-binding patterns in Gallus domesticus.

BACKGROUND: Although chicken oviduct is a useful model and target tissue for reproductive biology and transgenesis, little is known because of the highly specific hormonal regulation and the lack of fundamental researches, including lectin-binding activities and glycobiology. Because lectin is attached to secreted glycoproteins, we hypothesized that lectin could be bound to secretory egg-white proteins, and played a crucial role in the generation of egg-white protein in the oviduct. Hence, the purpose of this study was to investigate the structural, histological and lectin-binding characteristics of the chicken oviductal magnum from juvenile and adult hens. METHODS: The oviductal magnums from juvenile and adult hens were prepared for ultrastructural analysis, qRT-PCR and immunostaining. Immunohistochemistry of anti-ovalbumin, anti-ESR1 and anti-PGR, and mRNA expression of egg-white genes and steroid hormone receptor genes were evaluated. Lectin histochemical staining was also conducted in juvenile and adult oviductal magnum tissues. RESULTS: The ultrastructural analysis showed that ciliated cells were rarely developed on luminal surface in juvenile magnum, but not tubular gland cells. In adult magnum, two types of epithelium and three types of tubular gland cells were observed. qRT-PCR analysis showed that egg-white genes were highly expressed in adult oviduct compared with the juvenile. However, mRNA expressions of ESR1 and PGR were considerably higher in juvenile oviduct than adult (P < 0.05). The immunohistochemical analysis showed that anti-ovalbumin antibody was detected in adult oviduct not in juvenile, unlikely anti-ESR1 and anti-PGR antibodies that were stained in both oviducts. In histological analysis, Toluidine blue was stained in juvenile and adult oviductal epithelia, and adult tubular glands located in the outer layer of oviductal magnum. In contrast, PAS was positive only in adult oviductal tubular gland. Lectins were selectively bound to oviductal epithelium, stroma, and tubular gland cells. Particularly, lectin-ConA and WGA were bound to electron-dense secretory granules in tubular gland. CONCLUSIONS: The observation of ultrastructural analysis, mRNA expression, immunohistochemistry and lectin staining showed structural and physiological characterization of juvenile and adult oviductal magnum. Consequently, oviduct study could be helped to in vitro culture of chicken oviductal cells, to develop epithelial or tubular gland cell-specific markers, and to understand female reproductive biology and endocrinology.

Distribution of chitinases and characterization of two chitinolytic enzymes from one-year-old Korean Ginseng (Panax ginseng C.A. Meyer) roots.

We report the tissue-specific distribution of chitinolytic activity in Korean ginseng root and characterize two 31-kDa chitinolytic enzymes. These two enzymes (SBF1 and SBF2) were purified 70- and 81-fold with yields of 0.75 and 1.25%, respectively, and exhibited optimal pH and temperature ranges of 5.0-5.5 and 40-50(o)C. With [(3)H]-chitin as a substrate, K(m) and V(max) values of SBF1 were 4.6 mM and 220 mmol/mg-protein/h, respectively, while those of SBF2 were 7.14 mM and 287 mmol/mg-protein/h. The purified enzymes showed markedly less activity with p-nitrophenyl-N-acetylglucosaminide and fluorescent 4-methylumbelliferyl glycosides of D-N-acetylglucosamine oligomers than with [(3)H]-chitin. End-product inhibition of both enzymes demonstrated that both are endochitinases with different N-acetylglucosaminidase activity. Furthermore, the NH(2)-terminal sequence of SBF1 showed a high degree of homology with other plant chitinases whereas the NH(2)-terminal amino acid of SBF2 was blocked. [BMB reports 2010; 43(11):726-731].

CpG methylation modulates tissue-specific expression of a transgene in chickens.

The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G(1) generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G(3) generation. Based on serum biochemistry, there were no significant physiological differences between G(3) homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G(3) chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.

Identification of the major proteins produced by cultured germline stem cells in chicken.

Although chicken spermatogonial stem cells (SCs) are important in spermatogenesis and transgenic research, little is known about these cells. Recently, our group constructed an in vitro culture system to establish germline stem cells (GSCs). To examine the mechanism of chicken spermatogonial SC development, we constructed a proteome map of GSCs from 4-week-old chicken testes. Soluble extracts of the GSCs were fractionated by 2-dimensional gel electrophoresis (pH 4-7). Several protein spots, including those that displayed significantly high levels, were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Of the 82/250 GSC spots examined, 56 yielded mass spectra that matched avian proteins found in the on-line databases. All of the identified proteins were classified into functional groups. This type of proteome map is an important resource for research on spermatogenesis and transgenesis.

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken.

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.

ChickGCE: a novel germ cell EST database for studying the early developmental stage in chickens.

We established a database to study germ cells during the early developmental stage in the chicken. The ChickGCE database provides integrated expressed sequence tag (EST) data from chicken testis, ovary, embryonic gonads, and primordial germ cells. We gathered data on 10,294 ESTs from approximately 1000 embryonic gonads, and we experimentally determined 10,851 ESTs from primordial germ cells purified from 7955 embryonic gonads by magnetically activated cell sorting. The EST testis and ovary datasets were retrieved from the public database of The Institute for Genomic Research (TIGR). The EST data were clustered and assembled into unique sequences, contigs, and singletons. The ChickGCE database provides functional annotation, identification, and putative embryonic germ-cell-specific novel transcripts based on the Gene Ontology database, as well as statistical analyses of expression patterns and pair-wise comparisons of two types of tissue- and germ-cell-specific alternative splicing events in the chicken. The new database is accessible online and queries can be answered using several search options, including tissue database searches, keywords, clone IDs, expected values, and BLAST search scores.

Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells.

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.

Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen.

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V(H) and V(L) genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l(-1). The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V(L) genes suggested that the gene conversion with (V)lambda pseudogenes might contribute to the diversification of V(L) genes in chickens.