Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk

We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors.     

Production of scFv-Fc fusion protein using genetically manipulated quails

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.     

Production of anti-prion scFv-Fc fusion proteins by recombinant animal cells

We constructed a replication-defective retroviral vector plasmid for the expression of a single-chain antibody fragment (scFv), derived from a chicken anti-human prion protein monoclonal antibody, fused with the Fc region of human IgG1. CHO-K1 and NS-1 cells were transformed with the viral vector pseudotyped with vesicular stomatitis virus G protein (VSV-G), and scFv-Fc producer clones were established. Among the established clones, CHO-2A9 cells produced a large amount of the product with an antibody-like dimerized structure in serum-free culture that facilitated the purification of scFv-Fc. The scFv-Fc specifically recognized the epitope sequence of prion protein in solid-phase enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The injection test into quails revealed that the scFv became more stable in vivo by fusion with the Fc region. The scFv-Fc will be a useful tool for the detection of mammalian prion proteins.     

Localization of primordial germ cells or their precursors in stage X blastoderm of chickens and their ability to differentiate into functional gametes in opposite-sex recipient gonads

This study was performed to determine the distribution of primordial germ cells and their precursors in stage X blastoderm of chickens. The blastoderm (Barred Plymouth Rock chickens) isolated from the yolk was separated into three portions: the central disc, the marginal zone and the area opaca. The dissociated blastodermal cells derived from the central disc, marginal zone and area opaca were transferred into a recipient blastoderm (White Leghorn chicken) from which a cell cluster was removed from the centre of the central disc. The manipulated embryos were cultured in host eggshells until hatching. The chicks were raised until sexual maturity and test mated with Barred Plymouth Rock chickens to assess the donor cell contribution to the recipient germline. Germline chimaeric chickens were produced efficiently (46.7%, 7/15) when the blastodermal cells derived from the central disc were transferred into recipient embryos of the same sex, whereas no germline chimaeric chickens were produced when the blastodermal cells derived from the marginal zone or area opaca were transferred into recipient embryos of the same sex (0/12). Germline chimaeric chickens were also produced by transfer of blastodermal cells derived from the central disc (6.7%, 1/15), marginal zone (10.0%, 1/10) or area opaca (11.1%, 1/9) into recipient embryos of the opposite sex. It is concluded that primordial germ cells are induced during or shortly after stage X and that the cells derived from the central disc have the highest potential to give rise to germ cells. Cells derived from the marginal zone and area opaca can also give rise to germ cells, although the frequency is low.     

Effect of layer management on carotene value in eggs

Chromatographic-spectrophotometric method was used to determine the carotene content in the egg yolk of naturally raised chickens and in the eggs produced on poultry farms (industrial production) for the winter and spring periods. The aim of the study was to find out if the industrial production of eggs causes changes in the carotene content of the egg yolk. Distinct differences were recorded in the carotene content in the egg yolk of eggs obtained from private producers and of those produced on poultry farms, for both periods. Considerable differences were recorded for the spring period: the carotene content in the yolk of eggs laid in natural environment, by chickens fed natural food, was 290% higher than that in the yolk of industrially produced eggs. The difference for the winter period was 152%.     

A rapid strip immunoblot assay for the specific detection of Salmonella enteritidis infection in chickens

A rapid strip immunoblot assay (RSIA) was developed using recombinant SEF14 antigen. The rSEF14-RSIA was very specific in detecting antibodies to Salmonella enteritidis in chickens. When serum samples obtained from groups of chickens (N = 5) inoculated with six different Salmonella serovars were tested in rSEF14-RSIA, only serum samples obtained from S. enteritidis inoculated birds reacted with rSEF14 antigen except for a group of chickens that had been inoculated with S. dublin. To assess the sensitivity of the rSEF14-RSIA, groups of chickens were inoculated with either 10(4) cfu or 10(10) cfu of S. enteritidis and the serum and egg yolk were analyzed for SEF14 antibodies. By 1 week after infection 66-78% of chickens were found positive for SEF14 antibodies in the serum and the number of positive birds increased subsequently to 89-100%. The S. enteritidis specific antibodies appeared as early as 6 days after infection in the egg yolk of infected chickens. The antibodies to SEF14 in both the serum and egg yolk persisted for at least 7 weeks after infection in a significant proportion of chickens. Our results suggest that rSEF14-RSIA can be a practical and efficient screening test for identifying S. enteritidis infected chickens.     

人组织型纤溶酶原激活剂转基因功能鸡蛋的研究及其活性分析

为探索人组织型纤溶酶原激活剂(t-PA)在鸡卵黄中积淀的可行性,开发具有抗心血管疾病的功能性鸡蛋,应用分子生物学技术分别构建含有人t-PA 基因和t-PA 与鸡卵黄高磷蛋白融合基因的真核表达质粒pcDNA3-tPA 和pPhosvitin-tPA,脂质体包裹后分别注射于初产蛋鸡的肝脏,Western blotting 和ELISA 检测t-PA 基因在鸡肝脏中的表达和在卵黄中的积淀情况,琼脂糖平板溶圈法检测期其活性。结果显示,注射重组质粒后7d,卵黄中有分子质量为63kD 的t-PA 积淀,表达可持续5 周,高峰表达量分别为39.16mg/L 和53.92mg/L;琼脂糖平板溶圈法检测其活性表明,卵黄中的t-PA 具有激活组织纤溶酶的活性。实验证明了外源基因表达产物在卵黄中积淀这一途径的可行性。     

禽蛋黄过敏原Gal d 6的重组表达、纯化鉴定

目的:建立表达蛋黄Gal d 6 重组蛋白工程菌,并鉴定重组Gal d6蛋白免疫活性。方法:直接合成Gal d 6 DNA,与pET-28a构建重组质粒,转化E.coli BL21经IPTG诱导表达;优化表达条件制备重组Gal d 6蛋白,经镍离子亲和层析柱纯化得到纯品蛋白;纯化Gal d6蛋白经间接ELISA及western blot鉴定Gal d 6蛋白免疫活性。结果:经DNA测序、SDS-PAGE、禽蛋过敏血清鉴定表达Gal d 6蛋白具有免疫活性,经镍柱亲和层析获得单一条带。该工程菌最佳表达条件为37 ℃,0.5 mmol/L IPTG,2 h,收获量每升菌液可收获重组蛋白约9.1 mg。重组蛋白与禽蛋过敏血清具有良好反应性。结论:建立稳定表达具有免疫活性的Gal d 6蛋白的工程菌株。     

Limited enzymatic proteolysis increases the level of incorporation of canola proteins into mayonnaise

An alkaline extract of canola meal was hydrolyzed using a protease (Proleather FG-F) for 2.5 and 10 min to obtain protein hydrolysates with 7% and 14% degree of hydrolysis (DH), respectively. The protein hydrolysates were used to replace up to 50% (w/w) of egg yolk in a model mayonnaise preparation and the effects on physicochemical properties determined. Unhydrolyzed canola proteins could only be incorporated into mayonnaise up to a maximum 15% (w/w) substitution of egg yolk without emulsion breakdown. At 7% DH, the canola protein could be used to substitute up to 20% (w/w) of egg yolk, while at 14% DH the maximum level of substitution was 50% (w/w). Increased level of canola protein products reduced the whiteness and increased the reddish-brown colour of mayonnaise. Mayonnaise that was stabilized by only egg yolk had the least particle size and highest viscosity when compared to emulsions stabilized by canola proteins. Stability studies showed that most of the mayonnaise preparations retained their physicochemical properties after storage for 4 weeks at 4 °C. The results suggest that limited protein hydrolysis can be used to increase the level of incorporation of globular proteins into mayonnaise preparations.Industrial relevanceModifications to the native structures of plant proteins remain a viable industrial option for improving their functionality in food systems. Therefore, the process described in this work could promote utilization of enzymatically modified canola proteins as suitable ingredients in the formulation of food emulsions.     

Multiplication and motility of Salmonella enterica serovars enteritidis, infantis, and montevideo in in vitro contamination models of eggs

The invasive ability of Salmonella enterica serovars Enteritidis, Infantis, and Montevideo in eggs was examined. Strains of these serovars originating from egg contents, laying chicken houses, and human patients were experimentally inoculated (0.1-ml dose containing 78 to 178 cells) onto the vitelline membrane of eggs collected from specific-pathogen-free chickens and incubated at 25 degrees C. The test strains were detected in 25 of 138 yolk contents by day 6, indicating the penetration of Salmonella organisms through the vitelline membrane. There were no significant differences in overall rates of penetration between serovars. The organisms were also detected in the albumen from 125 of 138 eggs tested by day 6. Growth to more than 10(6) CFU/ml was observed in 48 of the 125 albumen samples. An inoculum of 1000 Salmonella cells was added to 15 ml of albumen at the edge of a petri plate. A 10-mm-diameter cylindrical well, the bottom of which was sealed with a polycarbonate membrane with 3.0-microm pores, was filled with egg yolk and placed into the albumen at the center of the dish, which was maintained at 25 degrees C. Experiments were performed in triplicate with each strain. Salmonella organisms in all the albumen samples were detected by day 11. However, motility of the organisms toward the yolk was observed in only two dishes inoculated with the Salmonella Enteritidis strain from a human patient and in one dish inoculated with the Salmonella Infantis strain from liquid egg. The albumen samples obtained from the dishes inoculated with the Salmonella Enteritidis strain had high numbers of bacteria (>10(8) CFU/ml). The present study suggests that Salmonella organisms in egg albumen are unlikely to actively move toward the yolk, although depositionon or near the vitelline membrane can be advantageous for proliferation.     

Clarification of Spray-Dried Egg Yolk Suspensions and Solubilization of Proteins from Lipoproteins

The separation of proteins and lipids from spray-dried egg yolk was attempted using commerical enzymes and mold enzymes. Good solubilization of protein from a spray-dried egg yolk suspension (clarification of the suspension), could not be obtained with purified commercial enzymes (protease, lipase and phospholipase), but three crude enzymes extracted from molds and one commercial enzyme produced good results. Among them, Newlase F (crude preparation, derived from the genus Rhizopus) successfully clarified the spray-dried egg yolk suspension (pH 4.7, 40°C, 5–8 hr), thereby solubilizing about 85% of the total protein in the whole spray-dried egg yolk.     

Immunogenic and allergenic potentials of natural and recombinant innocuous proteins

A new aspect of protein immunogenic and allergenic properties has become important recently, when there is a higher chance that our immune system will be exposed to novel protein antigens and/or familiar protein antigens with an unprecedented high frequency and large amount. These proteins are innocuous, nontoxic, and noninvasive by themselves, and include various natural proteins from the environment and recombinant proteins from industry. The technical term allergenic has been used for such proteins and their abilities to induce specific IgE production and to cross-link IgE/Fc epsilonRI on the surface of mast cells and basophiles have been recognized. As for the environmental proteins, some physicochemical properties (solubility, stability, and permeability across a mucosal epithelium) of the proteins indirectly play important roles in their allergenic potential because they do not originate from invasive pathogens as vehicles. Indeed, several lines of experimental evidences have been accumulated indicating that all proteins are absorbed across mucosal epithelia by transcellular transport and/or through interstitial spaces among the epithelial cells but not at equal levels. Some animal models have been established for natural sensitization to some allergenic proteins by feeding or intragastric administration without an adjuvant and, in a few cases, some symptoms resembling human allergy and even anaphylaxis have been induced by oral challenge with the proteins. Sometimes, even to self-proteins, the immunogenic or allergenic potential is given by post-translational modifications and possibly by unknown structural/conformational alterations, when they are exogenous self-proteins, such as recombinant human proteins for drug use. Despite the accumulation of knowledge and the progress in analytical technology on protein allergenicity, it is still crucial to predict the allergenic potential of novel and unused proteins. However, some animal models are applicable for assessing the relative allergenic potential of processed proteins in comparison with that of native proteins in preclinical studies.     

DISC GEL ELECTROPHORESIS OF PROTEINS IN NATIVE AND HEAT-TREATED ALBUMEN, YOLK, AND CENTRIFUGED WHOLE EGG

SUMMARY– Polyacrylamide disc gel electrophoresis was an effective technique for resolving the proteins of albumen, yolk, and centrifuged whole egg into distinct bands. Albumen proteins including ovalbumins, conalbumins, and globulins were separated into 12 bands. Ovomucoid could not be detected and lysozyme did not migrate into the gel. With yolk, 19 bands including livetins and lipovitellins but not low-density lipoproteins were formed. When centrifuged whole egg was subjected to electrophoresis, livetin and albumen protein bands were detected, but no evidence was obtained for the formation of new protein complexes. Alterations of gel patterns were observed when albumen, yolk, and centrifuged whole egg were heated to pasteurization temperatures of 61.7°C and above.     

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.     

The role of Tween in inhibiting heat-induced destabilization of yolk-based emulsions

The process of heat-induced destabilization of yolk-based emulsions and the role of Tween addition in inhibiting droplet aggregation/coalescence in the thermally treated emulsions were investigated. The aim of the study was to understand the mechanism behind yolk emulsion destabilization during the application of processes such as pasteurization/sterilization and/or cooking. Data on emulsion particle size distribution were combined with results on yolk protein adsorption to clarify the role of the unadsorbed yolk protein fraction in the destabilization of the thermally treated emulsion. Surface tension measurements were also conducted to investigate yolk protein–Tween interactions at the air/water interface and their effect on emulsion stability. The presence in the emulsion continuous phase of unadsorbed yolk protein is crucial for the thermal destabilization of the system. Tween addition inhibits droplet flocculation/coalescence phenomena by shielding the reactive groups of protein molecules adsorbed at the droplet surfaces and those of unadsorbed proteins in the emulsion continuous phase which become available for interaction following heating and protein denaturation.     

基于质谱检测转基因生物外源蛋白质的消化稳定性

基于无标记定量质谱检测技术建立了一种新的转基因生物外源蛋白质消化稳定性评价方法。以牛β-乳球蛋白、牛血清白蛋白和大豆胰蛋白酶抑制剂为标准蛋白质,经模拟人体胃/肠消化液消化后进行凝胶电泳分离,切取目标蛋白质条带进行酶切,提取肽段进行纳升液相色谱-电喷雾串联质谱分析。基于Mascot数据库检索鉴定目标蛋白质。计算各消化时间点蛋白质匹配肽段数与消化前蛋白质匹配肽段数的比值,当比值≤0.50时判断为目标蛋白质在该时间段内已消化。将此方法应用于转基因抗虫水稻“华恢1号”外源蛋白Cry1Ab/1Ac的消化稳定性分析,结果显示在模拟胃液中消化2 min时,比值下降到0.50以下,在模拟肠液中消化15 s后比值下降到0.50以下,表明该蛋白在胃/肠消化液中具有消化不稳定性。     

Simple purification methods for an alphagalactose-specific antibody from chicken eggs

For many applications avian antibody from egg yolk (IgY) offers advantages over the well-known mammalian antibodies. Different experimental techniques for the purification of IgY from chickens immunized with an alphagalactose-containing antigen (alphaGal-trisaccharide) were compared. These included ammonium sulfate precipitation, filtration with diatomaceous earth, treatment with deoxycholate, and thiophilic and affinity chromatography. Samples were tested for overall purity, protein and lipid content, and specific activity. Evaluated on the basis of these results and the simplicity of the process, the favored purification method is ammonium sulfate precipitation of diluted egg yolk directly followed by affinity chromatography. The high lipid content of IgY preparations is greatly reduced by either thiophilic or affinity chromatography. Affinity purification of ammonium sulfate precipitated material resulted in anti-alphaGal-trisaccharide IgY preparations with approximately 1% of the original protein content but approximately 100-fold higher specific activity for the alphaGal-trisaccharide epitope.     

Food allergy—towards predictive testing for novel foods

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category includes foods produced using novel processes, genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed crossreactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain), and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.     

Egg yolk protein and egg yolk phosvitin inhibit calcium, magnesium, and iron absorptions in rats

ABSTRACT:  Egg yolk decreases the absorption of iron. The effects of egg yolk protein and egg yolk phosvitin on the absorption of calcium, magnesium, and iron were investigated by in vivo studies. Male Wistar rats were fed purified diets containing casein, soy protein, or egg yolk protein for 14 d. The apparent absorptions of calcium, magnesium, and iron in the rats fed the yolk protein-based diet were lower than those in rats fed the casein- and soy protein-based diets. The apparent phosphorus absorption and the apparent protein digestibility in the yolk protein group were lower than those in the casein and soy protein groups. In the feces of the yolk protein group, serine comprised more than 30% of the amino acids. The addition of egg yolk phosvitin to the casein diets at levels of 1% and 2% (w/w) produced effects on calcium and magnesium absorptions similar to those produced by the diet containing yolk protein. The tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern suggested that phosphopeptide fragments having molecular masses of 28, 22, and 15 kDa were evident in the contents of the small intestine of the rats fed phosvitin diets. These results indicate that yolk protein, when compared with casein and soy protein, decreases calcium and magnesium absorption via the resistance of phosvitin to proteolytic action.