Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens

We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.     

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.     

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.     

Functional and heterologous expression of human protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 in zebrafish

Although membrane-associated proteins are related to many diseases and are important targets for drug discovery, their expression is often difficult in bacterial hosts such as Escherichia coli. To overcome this limitation, here, we focused on a novel host-vector system in zebrafish for the expression of human protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (hPOMGnT1) which is related to muscle-eye-brain disease. For the expression of hPOMGnT1, the vector pZex-EGFP-pXI-hPOMGnT1 was constructed and injected into fertilized eggs. Using this system, we demonstrated that recombinant hPOMGnT1 was successfully expressed in the whole bodies of zebrafish embryos.     

Solubility of Proteins from Quail (Coturnix coturnix japonica) Egg White as Affected by Agitation Time,pH, and Salt Concentration

The solubility of proteins from quail egg white subjected to different agitation times, pH, and NaCl at 25°C was evaluated in this study. The greatest solubility achieved was obtained in the 0.05 mol/L of NaCl, pH 10.0, and under agitation for 1 h. The lowest solubility was found in the NaCl 1.0 mol/L, pH 3.0, and agitated for 2 h. Under the test conditions, aqueous solutions of NaCl 1.0 mol/L, pH 3.0, and agitated for 2 h may provide the greatest extraction index of proteins from quail egg white because these conditions promoted the protein precipitation in a large extension.     

Comparative study on physicochemical and functional properties of egg powders from Japanese quail and white Leghorn chicken

Physicochemical and functional properties of freeze-dried egg powders (egg white, egg yolk, and whole egg) from Japanese quail and white Leghorn chicken were studied comparatively. All egg powders had protein content in the range of 91.13–97.03 g/100 g powder. The quail egg powder had higher mineral and essential amino acid contents, but lower fat content as compared to chicken egg powders (P < 0.05). Moreover, egg white powder from both quail and chicken presented higher total amino acids content than corresponding whole egg and egg yolk powders, respectively. Fourier transform infrared spectroscopic study revealed that β-sheet is the major secondary structure of all egg powders. Based on differential scanning calorimetry analysis, quail egg powders showed slightly lower denaturation temperatures than corresponding chicken egg powders (P < 0.05). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis study showed the slight difference in protein patterns of corresponding quail and chicken egg powders. The quail egg powders presented higher protein solubility than corresponding chicken egg powders at all pH tested. Furthermore, quail egg powders exhibited higher emulsion activity index and emulsion stability index with higher foam expansion and stability than the corresponding chicken egg powders. Therefore, Japanese quail egg powders could be used as an alternative to white Leghorn chicken egg in the preparation of foods and diets that require high protein content with positive health benefits.     

Response of laying Japanese quails to graded levels of essential amino acids profile with reduced dietary protein

The effect on egg production of graded levels of ideal amino acids, combined with reduced protein in the diet, was investigated in 312 laying quails aged 6–18 weeks. The quails were offered six diets, each of which contained one of three levels of amino acids (85, 100 and 115% of essential amino acids (EAAs)) together with 5% or without fishmeal (FM) (3 × 2 factorial design). Each diet was offered to 26 replicated groups of two quails each. Hen‐day and hen‐housed egg production did not differ as a result of EAA level, protein type or their interaction during the overall period of egg production. Egg weight improved linearly (P < 0.01) with increased EAA levels, while egg mass output per bird per day remained similar at the 100 and 115% EAA levels. Quails fed higher (100 and 115%) EAA levels had an improved feed conversion ratio (P < 0.01) compared to birds fed 85% EAA. The gain in body weight during the laying period was higher (P < 0.01) at the 100 or 115% than at 85% EAA levels The ratio of egg mass or egg mass and live weight gain, together, to protein intake improved (P < 0.01) linearly with a decrease in EAA levels in the diets, while better (P < 0.01) energy efficiency (EE, energy intake: egg mass) and net EE (energy intake: egg mass plus gain) was obtained in higher EAA levels (100 or 115%). Protein and energy efficiencies remained similar due to protein type or interaction. Shape index, albumen index, yolk index, yolk colour and relative shell weight did not differ due to EAA levels, protein type or their interaction. Eggs laid from quails fed diets with 100% EAAs without FM and 115% EAAs with or without FM had higher shell thickness than those on 85% EAAs irrespective of protein type. The retention of nitrogen and calcium retention was higher (P < 0.01) at the 115% EAA level. The results indicated a dietary level of 100% EAA (185 g kg^?1 crude protein (CP)) with 12.13 MJ kg^?1 was suitable for laying quails of 6–18 weeks of age. Copyright © 2007 Society of Chemical Industry     

EFFECTS OF HEAT TREATMENT AND PEPSIN DIGESTION ON TRYPSIN INHIBITORY ACTIVITY AND OVOMUCOID ANTIGENICITY OF JAPANESE QUAIL EGG WHITE

Changes in trypsin inhibitory activity and ovomucoid antigenicity in the egg white of Japanese quail egg were observed after subjecting the quail egg to boiling, saúteing and microwave irradiation. Boiling for 10 min caused about 50% loss of the initial trypsin inhibitory activity. With respect to antigenicity, the boiling caused an initial increase followed by a drop to the original level. Saúteing for 3 min resulted in about 90% loss of the inhibitory activity and about 75% loss of the initial antigenicity. Microwave irradiation for 90 s resulted in about 85% loss of the inhibitory activity and about 80% loss of the initial antigenicity. After pepsin digestion of the egg white boiled for 10 min, 100% of the starting trypsin inhibitory activity was retained even after 24 h digestion, while the antigenicity decreased to about 10% of the initial activity. These results suggest that about 50% of the original trypsin inhibitory and 10% of the original antigenic activity remain after a 24 h pepsin digestion of the egg white boiled for 10 min. Thus, we concluded that intact ovomucoid or ovomucoid degradation products with some trypsin inhibitory activity and antigenicity were introduced into the small intestine after ingestion of the cooked egg white of Japanese quail.     

鹌鹑蛋功能特性及其影响因素

测定了鹌鹑蛋乳化性、起泡性、持水性等功能特性,并探讨了蔗糖、温度、pH值、氯化钠等因素对蛋白起泡性及其稳定性和蛋黄乳化性的影响。结果表明,鹌鹑蛋白的起泡性为7.33%,泡沫稳定性为63.49%,乳化性为41.51%,持水性约17.14%;鹌鹑蛋黄的起泡性为10%,泡沫稳定性为59.73%,乳化性为46.82%;鹌鹑全蛋液的起泡性为11%,泡沫稳定性为63.95%,乳化性为43.99%,持水性约18.28%。蔗糖能在一定范围内提高泡沫稳定性,适当加热可改善蛋白起泡性,蛋白起泡性随NaCl增加而有较大幅度提高。鹌鹑蛋黄乳化性随蔗糖浓度增大而提高,10～60℃内随温度升高而提高,加入氯化钠能明显提高蛋黄乳化性。pH值对蛋白起泡性和泡沫稳定性、蛋黄乳化性的影响均呈动态变化。     

基于iTRAQ技术的鸡蛋清三种干燥方式差异蛋白质组分析

为探究真空冷冻干燥(vacuum freeze-drying, FD)、喷雾干燥(spray-drying, SD)和微波真空冷冻干燥(microwave vacuum freeze-drying, MFD)3种干燥方式对蛋清蛋白的差异,利用iTRAQ技术对三者进行蛋白质组差异分析,在得到的157种具有定量信息的蛋清蛋白中,鉴定出87种差异蛋白。MFD vs FD组上调蛋白质数量为46个,占差异蛋白质总数的67.6%。KEGG富集显示,MFD vs FD组和MFD vs SD共有2个基因通路显著富集,分别为折叠和降解、运输和分解代谢。此外,在差异蛋白中筛选出4种蛋白质在干燥过程中丰度变化显著,分别为黏蛋白、血清白蛋白、卵清蛋白相关蛋白Y亚型X1和卵白蛋白相关蛋白Y。该研究首次采用蛋白质组学的方法分析不同干燥方式处理后的鸡蛋清差异蛋白的表达变化,为今后生产和开发高品质蛋清粉及功能性蛋制品提供参考依据。     

Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated gamma-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.     

Effect of pasteurisation on the chemical composition of liquid whole egg. I. Development of a scheme for the fractionation of the proteins of whole egg

The proteins of whole egg may be separated into soluble and insoluble portions by dialysis against glycine solution followed by centrifugation at a high speed. The soluble proteins can then be resolved into at least twelve fractions by ion-exchange chromatography on diethylaminoethyl-cellulose, using stepwise elution with dilute glycine-phosphate buffer solutions containing increasing concentrations of sodium chloride. Further examination has shown that most of the fractions contain more than one protein, and some tentative identifications have been made. The insoluble proteins can be dissolved in a relatively strong phosphate buffer and fractionated on diethylaminoethyl-cellulose in a similar way. Separate examinations of white and yolk indicated that the soluble proteins of whole egg are predominantly the same as those of the white, and also showed certain differences between whole egg and the separated white and yolk that could be attributed to some form of protein association in mixed whole egg. An initial experiment has suggested that the method described will reveal differences between raw and pasteurised egg.     

Effects of dietary taurine on egg production,egg quality and cholesterol levels in Japanese quail

BACKGROUND: Taurine is a semi‐essential amino acid and has many biological properties. The objective of this study was to determine the effect of dietary supplementation with taurine on egg production, egg quality, and cholesterol level in serum and egg yolk of quails. A total of 108 quails aged 6 weeks were randomly allocated to three dietary treatments. Each treatment consisted of four replicates of nine quails. The diets were supplemented with 0, 100, and 500 mg kg^?1 of taurine for 8 weeks. RESULTS: Dietary 500 mg kg^?1 taurine significantly affected egg production rate and feed conversion ratio, but had no significant effects on body weight gain, feed consumption, or egg weight. Dietary taurine had no significant effect on egg quality parameters studied. Serum triglyceride concentration was reduced significantly with supplementation of taurine at 100 and 500 mg kg^?1. Egg yolk cholesterol content was reduced significantly, and the contents of serum taurine and egg yolk taurine were increased significantly with taurine supplementation at 500 mg kg^?1. CONCLUSION: Results of the present study indicated that adding 500 mg kg^?1 taurine reduced yolk cholesterol concentration and increased yolk taurine content without adverse effects on performance and egg quality of laying quails. Copyright © 2010 Society of Chemical Industry     

High-level expression of recombinant human nerve growth factor beta in milk of nontransgenic rabbits

The technology for the large-scale production of therapeutic recombinant proteins remains a challenge in the biopharmaceutical industry. In this study, we reported a nontransgenic approach to producing a large quantity of human nerve growth factor beta (hNGF-beta) in rabbit milk by employing a recombinant adenoviral expression system. After directly instilling hNGF-beta recombinant adenoviruses into rabbit mammary glands, a polypeptide with a molecular weight of 13.2 kDa was detected in rabbit milk. The maximal expression level of hNGF-beta reached 346 mug/ml. The biological activity of recombinant hNGF-beta was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. Our data suggest that instilling recombinant adenovirus directly into the mammary gland of mammals is an efficient approach to producing a large quantity of hNGF-beta.     

Modification of Egg White Proteins with Oleic Acid

Studies on the chemical modification of egg white with oleic acid (5-50 moles/50,000g of egg white protein) revealed that the reagent partitioned equally between supernatant and precipitate. The mole ratio of oleic acid to protein in solution at the 20 mole level of treatment was 14.6:1. Oleic acid did not selectively precipitate ovalbumin, conalbumin, or lysozyme. An increase in negative charge of proteins was observed in the chromatograms of treated egg white. No difference in molecular weights of treated egg white proteins was observed. The viscosity varied with the concentration of the reagent. In cooked, frozen, and thawed egg white water retaining index was 1.5-10X greater for treated than untreated material.     

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。     

On hen egg fractionation: applications of liquid chromatography to the isolation and the purification of hen egg white and egg yolk proteins

Liquid chromatography has been used as a means of egg protein analysis or as a method for the purification of egg proteins. Several Chromatographic methods, including gel permeation, ion-exchange, reversed-phase, hydrophobic interaction, and immobilized-ligand-affinity chromatography, have been carried out for the separation or the purification of egg yolk or egg white proteins. Ion-exchange chromatography appears to be the most frequently used method for protein isolation and it is the easiest to adapt to a process scale. From an analytical point of view reversed-phase chromatography is, at the moment, the recommended method for egg white analysis. Egg white has been fractionated more often by liquid chromatography than has egg yolk. Several Chromatographic methods have been developed on a laboratory scale, but the application of these techniques on an industrial scale remains limited.     

Improving Foaming Properties of Yolk-Contaminated Egg Albumen by Basic Soy Protein

ABSTRACT:  Yolk contamination of egg white is a common problem in the egg breaking industry. Foaming properties of egg white protein are affected by such contamination, but proteins of basic nature may restore the foaming properties of the yolk-contaminated egg white protein. The purpose of this study was to chemically modify a soy protein, that is, to esterify the acidic groups on the protein and to study the potential of such modified protein in improving foaming. We showed that the modification changed the isoelectric point of soy protein isolate (SPI) from 4.5 to about 10. Sonication was proven to be a very effective means to redisperse the methanol-denatured soy protein during reaction, as shown by the improved solubility profile. Such modified basic protein, that is, the sonicated-modified SPI (SMSPI), when added to the yolk-contaminated (at 0.4% level, as-is basis) egg white, gave significantly improved foaming properties. We have shown that the slight change in pH due to the addition of SMSPI was not the reason for improved foaming performance; instead, the modified protein itself was the main reason for such improvement. Addition of SMSPI increased the foaming performance of both pure egg white and yolk-contaminated egg white. SMSPI consistently performed better than the unmodified SPI for improving foaming. Addition of SMSPI (16%, based on dry egg white, and 1.6% based on liquid egg white) fully restored foam expansion and foam liquid stability of 0.4% yolk-contaminated egg white, and it even out-performed the foaming of pure white protein. Therefore, a feasible solution to restore the foaming properties of yolk-contaminated egg white has been identified. It is expected that such modified SPI can be used as an additive or ingredient in foaming formulation, especially when the egg white protein is suspected of lipid contamination.