The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform
Yunping Dai - One of the best experts on this subject based on the ideXlab platform.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned Cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four Transgenic Cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of Transgenic and non-Transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes Transgenic Cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.
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88 cell cycle synchronization of fibroblasts derived from Transgenic cloned Cattle ear skin effects of serum starvation roscovitine and contact inhibition
Reproduction Fertility and Development, 2007Co-Authors: Xiuzhu Sun, Shuhui Wang, Yuanliang Zhang, Yunping DaiAbstract:Cell cycle stage plays a critical role in somatic cell nuclear transfer (SCNT), and G0/G1 stage cells are preferred nuclear donors in attempts to produce cloned livestocks. Enhancement of survivability of cloned calves by roscovitine (the cyclin-dependent kinase-2 inhibitor) has been shown (Gibbons et al. 2002 Biol. Reprod. 66, 895–900). The purpose of this study was to evaluate the effects of serum starvation, roscovitine, and contact inhibition on cell cycle synchronization at the G0/G1 stage of Transgenic cloned bovine ear skin-derived fibroblasts. The cell line was established from a cloned calf expressing green fluorescent protein (GFP). Data were analyzed by using SAS (8.0) with ANOVA (SAS Institute, Inc., Cary, NC, USA). At passage 2-6, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) in T-25 culture flasks from immediately after subculture until monolayer cells reached 90% confluence at 39°C, under 5% CO2 in humidified air. Then cells grown in different flasks were randomly distributed to groups: Serum starvation (SS, culture medium changed to DMEM + 0.5% FBS), roscovitine (R, cultured in DMEM + 10% FBS + 15 µM roscovitine), and contact inhibition (CI, DMEM + 10% FBS). From Day 1 to Day 5 after treatment, 3 flasks of cells from each group were subjected to fixation and staining every day, followed by determination of cell cycle stage with Becton Dickinson FACScan (Kues et al. 2000 Biol. Reprod. 62, 412–419). At least 3 replicates were performed for each determination. The results showed that in the cycling fibroblasts (50–60% confluence), 59.29% of the cells were at the G0/G1 phase. For the SS group, the proportion of G0/G1 cells was significantly higher for treatment lasting 3 days, 4 days, and 5 days than for treatment lasting 1 day or 2 days (91.5%c, 91.7%c, and 93.5%c vs. 90.1%b and 88.8%a, respectively; P < 0.05). No statistical differences were observed among 3-day, 4-day, and 5-day treatments. For the R group, one-day treatment was significantly lower in synchronizing cells to the G0/G1 stage than that from Day 2 to Day 5 (86.51%a vs. 91.1%a, 90.1%a, 89.4%a, and 91.3%b, respectively; P < 0.05) during which similar rates of G0/G1 phase were observed. For the CI group, an increase of G0/G1 stage cells was found after 3 days of CI (from 89.4%a for Day 1 and 90.4%ab for Day 2, to 91.4%bc for Day 3; P < 0.05), which was similar to 4 days (91.6%bc) and 5 days (92.1%c) for the CI group. We also found that the efficiency of obtaining G0/G1 phase cells was lower when roscovitine was employed to synchronize the cell cycle than when the SS and CI methods were used (89.7%b vs. 91.1%a and 91.0%a, respectively; P < 0.05), after analysis of the most effective treatment duration of each group. Our data indicate that in attempts to harvest adequate G0/G1-stage cells for re-cloning of Transgenic Cattle from fibroblasts established from ear skin of a newborn Transgenic cloned calf, 3 days of treatment is enough regardless of the method (SS, R, or CI). Further research is needed to evaluate the developmental competence of embryos cloned from cells prepared by the abovementioned treatments. [a-c mean statistical differences; P < 0.05.]
Jianwu Wang - One of the best experts on this subject based on the ideXlab platform.
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A novel glycosylated anti-CD20 monoclonal antibody from Transgenic Cattle
Scientific Reports, 2018Co-Authors: Ran Zhang, Chenjun Tang, Sheng Hou, Fangrong Ding, Huaizu Guo, Jianwu Wang, Jianmin Zhao, Bo Tang, Lei Zhao, Haiping WangAbstract:The monoclonal antibody (mAb) against CD20 known as Rituxan is widely used to treat autoimmune diseases and lymphomas. However, further application of Rituxan faces challenges of high production cost, which limits its availability in developing countries. Here, we report a new approach for large production of a recombinant anti-CD20 mAb in the milk of Transgenic Cattle (at a yield of up to ~6.8 mg/mL), with ~80% recovery rate and >99% purity. Crystallography study showed that our recombinant mAb is structurally nearly identical to Rituxan with only minor differences in N-linked glycosylation pattern. Functional study showed that, while our mAb shared similar target-cell binding capacities and complement-dependent cytotoxicity with Rituxan, our product exhibited a higher binding affinity for FcγRIIIα and a greater antibody-dependent cellular cytotoxicity. Accordingly, our recombinant mAb demonstrated a superior efficacy over Rituxan against B-cell lymphomas in severe combined immunodeficiency mice. Taken together, our data supports Transgenic Cattle as a novel model for cost-competitive, large-scale production of therapeutic antibodies.
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Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of Transgenic cloned cows
PLoS ONE, 2017Co-Authors: Yuhang Wang, Susanne Lindquist, Fangrong Ding, Olle Hernell, Jianwu Wang, Ling Li, Wenjie Liu, Tao Wang, Jing Li, Yaofeng ZhaoAbstract:Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of Transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of Transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of Transgenic Cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.
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molecular characterization of transgene integration by next generation sequencing in Transgenic Cattle
PLOS ONE, 2012Co-Authors: Ran Zhang, Yinliang Yin, Yujun Zhang, Hongxia Zhu, Qin Gong, Jianwu WangAbstract:As the number of Transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize Cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in Transgenic species.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned Cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four Transgenic Cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of Transgenic and non-Transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes Transgenic Cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.
Hong Guo - One of the best experts on this subject based on the ideXlab platform.
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insights into the function of n 3 pufas in fat 1 Transgenic Cattle
Journal of Lipid Research, 2017Co-Authors: Xinfeng Liu, Zhuying Wei, Chunling Bai, Xiangbin Ding, Lei Cheng, Li Zhang, Hong GuoAbstract:The n-3 PUFAs have many beneficial effects on human health, including roles in immunity, neurodevelopment, and preventing cardiovascular disease. In this study, we established reliable model fat-1 Transgenic Cattle using Transgenic technology and performed a systematic investigation to examine the function of n-3 PUFAs. Our results showed that expression of the fat-1 gene improved several biochemical parameters related to liver function and to plasma glucose and plasma lipid metabolism. Results of global gene and plasma protein expression analysis showed that 310 genes and 13 plasma proteins differed significantly in the blood of fat-1 Transgenic Cattle compared with WT Cattle, reflecting their regulatory roles in the immune and cardiovascular systems. Finally, changes in the gut microflora were also noted in the fat-1 Transgenic Cattle, suggesting novel roles for n-3 PUFAs in the metabolism of glucose and lipids, as well as anti-stress properties. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the role of n-3 PUFAs using models such as fat-1 Transgenic Cattle. This study provides novel insights into the regulatory mechanism of fat-1 in the immune and cardiovascular systems, as well as its anti-stress role.
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comparative muscle proteomics phosphoproteomics analysis provides new insight for the biosafety evaluation of fat 1 Transgenic Cattle
Transgenic Research, 2017Co-Authors: Xiangbo Xin, Xinfeng Liu, Xiangbin Ding, Shuping Yang, Congfei Jin, Hong GuoAbstract:The biosafety of fat-1 Transgenic Cattle has been a focus of our studies since the first fat-1 Transgenic cow was born. In this study, we used tandem mass tag labeling, TiO2 enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC-MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 Transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 Transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 Transgenic Cattle.
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microarray analysis of the gene expression profile and lipid metabolism in fat 1 Transgenic Cattle
PLOS ONE, 2015Co-Authors: Xinfeng Liu, Zhuying Wei, Chunling Bai, Xiangbin Ding, Hong GuoAbstract:Long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) are beneficial for human health. However, humans and mammals are unable to synthesize n-3 PUFAs because they lack the n-3 desaturase gene fat-1 and must therefore obtain this type of fatty acid through their diet. Through the production of fat-1 Transgenic animals, it is possible to obtain animal products that are rich in n-3 PUFAs, such as meat and milk. The aim of this study was to analyze the gene expression profile and the mechanism of lipid metabolism in fat-1 Transgenic Cattle and to accumulate important basic data that are required to obtain more efficient fat-1 Transgenic Cattle. Transcriptome profiling of fat-1 Transgenic and wild-type Cattle identified differentially expressed genes that are involved in 90 biological pathways, eight pathways of which were related to lipid metabolism processes 36 genes of which were related to lipid metabolism. This analysis also identified 11 significantly enriched genes that were involved in the peroxisome proliferator-activated receptor signaling pathway. These findings were verified by quantitative polymerase chain reaction. The information obtained in this study indicated that the introduction of an exogenous fat-1 gene into Cattle affects the gene expression profile and the process of lipid metabolism in these animals. These results may provide important insights into how an exogenous fat-1 gene synthesizes n-3 PUFAs in Transgenic Cattle and other mammals.
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fat 1 Transgenic Cattle as a model to study the function of ω 3 fatty acids
Lipids in Health and Disease, 2011Co-Authors: Tao Guo, Xin F Liu, Xiang B Ding, Fei F Yang, Yong W Nie, Hong GuoAbstract:ω-3 polyunsaturated fatty acids have been shown to play an important role in health. Enriched with ω-3 polyunsaturated fatty acids modulate expression of a number of genes with such broad functions as cell proliferation, growth and apoptosis and cell signaling and transduction, these effects, seem to regulate coronary artery disease, hypertension, atherosclerosis, psychiatric disorders and various cancer. In this context, fat-1 Transgenic Cattle was designed to convert ω-6 to ω-3 fatty acids could form an ideal model to study the effect of ω-3 fatty acids on the above functions. This study focuses on the total genomic difference of gene expression between fat-1 Transgenic Cattle and wild-type using cDNA microarrays, several genes were found to be overexpressed or suppressed in Transgenic Cattle relative to wild-type, these discrepancy genes related with lipid metabolism, immunity, inflammation nervous development and fertility.
Lei Zhang - One of the best experts on this subject based on the ideXlab platform.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned Cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four Transgenic Cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of Transgenic and non-Transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes Transgenic Cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.
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310 marker gene excision in Transgenic cloned somatic cells and effects on recloned embryo development in Cattle
Reproduction Fertility and Development, 2009Co-Authors: Shaohua Wang, Lei Zhang, Xiuzhu Sun, F R Ding, Kun Zhang, R Zhao, B Tang, F L Gao, H P Wang, Lili WangAbstract:Recently, it has become common to produce Transgenic Cattle by somatic cell nuclear transfer. As a major step, donor cells are transfected with expression vectors that contain not only the target gene but also marker genes such as neomycin resistant gene (neoR) used for subsequent cell screening. However, such markers in Transgenic animals may be undesirable for further researches (Kuroiwa et al. 2004 Nature Genetics 36, 775–780). Therefore, the aim of this study was to excise the marker gene (neoR) in Transgenic Cattle by using transiently expressed CRE recombinase. Initially, the presence of loxP sites flanking neoR was confirmed in fibroblast cells derived from human lysozyme Transgenic cloned Cattle. By using cassettes from plasmids PBS185, pIRES-EGFP and pIREShyg3, IRES-EGFP element was inserted downstream of the CRE coding region, and the co-expression vector, which permits translation of GFP and CRE from one messenger RNA, was constructed. Then Transgenic fibroblast cells were transfected by the CRE expression vector in circular format by Lipofectamine™ 2000 and screened by FACS after 48 h. GFP positive cells were selected to culture for 7 to 9 days, and removal of neoR was confirmed by PCR with 2000 to 3000 cells from each colony and the remaining cells used as donors for recloning. Then cleavage and blastocyst rate were evaluated; neoR excision was identified by single blastocyst PCR. Some blastocysts were harvested for differential staining and some used to conduct embryo transfer to evaluate in vitro and in vivo developmental ability. Data were analyzed using SAS (version 9.1) with one-way ANOVA. All experiments were replicated at least three times. We confirmed that loxP sites are correctly located at two sides of neoR. After transfection, neoR was removed from Transgenic cloned fibroblast cells and cre was found to be expressed transiently. Additionally, neoR excision was confirmed by single blastocyst PCR. Furthermore, no significant difference was found on cleavage (P = 0.15) and blastocyst rate (P = 0.73) between cloned embryos when using neoR free donors and neoR included donors. The differential staining study showed similar numbers in terms of total cell number (P = 0.69) and the ratio of ICM to total cell number (P = 0.96). After embryo transfer, successful establishment of pregnancies were observed and pregnancy rate on Day 90 and Day 120 (Day 1 is the 1st day after embryo transfer) are similar with control (P > 0.05). In conclusion, we successfully excised resistant marker gene from Transgenic cloned Cattle by transient expression of CRE recombinase and we expect it will benefit bioreactor and animal Transgenic research in the future.
Bin Yang - One of the best experts on this subject based on the ideXlab platform.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned Cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four Transgenic Cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of Transgenic and non-Transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes Transgenic Cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.
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characterization of bioactive recombinant human lysozyme expressed in milk of cloned Transgenic Cattle
PLOS ONE, 2011Co-Authors: Bin Yang, Jianwu Wang, Jianmin Zhao, Bo Tang, Yufang Liu, Chengdong Guo, Penghua Yang, Lei Zhang, Yunping DaiAbstract:Background There is great potential for using Transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.