[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
[Objective] The aim was to study whether recombinant Human Lactoferrin has toxic effect on Primary Hepatocyte and Nephrocyte Cell of rat to provide reference for further safety evaluation.[Method] Recombinant Human La...[Objective] The aim was to study whether recombinant Human Lactoferrin has toxic effect on Primary Hepatocyte and Nephrocyte Cell of rat to provide reference for further safety evaluation.[Method] Recombinant Human Lactoferrin and its digested products were taken as tested compound,cow Lactoferrin was used for contrast.Primary Hepatocyte and Nephrocyte Cell of rat were cultured and IC50 values were tested by MTT,and cytotoxic dose-response relationship was tested.[Result]Target toxicity was not found from recombinant Human Lactoferrin on hepatocytes and nephrocytes,in accordance with sub-chronic toxicity test.[Conclusion] This study is of reference value for further safety evaluation of recombinant Human Lactoferrin and safety of evaluation method of GM food.展开更多
Human lactoferrin(HLF)belongs to the transferrin family and serves as a crucial component of the innate im-mune system.Traditionally,lactoferrin is primarily extracted from whey.However,this method is constrained by l...Human lactoferrin(HLF)belongs to the transferrin family and serves as a crucial component of the innate im-mune system.Traditionally,lactoferrin is primarily extracted from whey.However,this method is constrained by low efficiency and high cost.Microbial cell factories have emerged as an important alternative solution to address this issue.This study aimed to achieve HLF expression by evaluating two host strains of Aspergillus niger,along with suitable promoters and signal peptides.Subsequently,we identified an appropriate glucoamylase(GlaA)fusion fragment to enhance HLF expression and secretion.The knockout of the proteases PepA and PepB reduced HLF degradation after secretion.Subsequently,a multi-gene integration strategy was employed to integrate HLF into three highly expressed loci and two protease loci,resulting in a yield of 54.3 mg/L.The knockout of the gene racA that regulates the polar growth of mycelia increased the branching frequency of A.niger hyphae,leading to a yield of 97.7 mg/L.Finally,we achieved a lactoferrin yield of 1027.1 mg/L through fermentation optimization in a 5-L bioreactor.The strategies adopted in this study provides a basis for the application of HLF in A.niger.展开更多
Human lactoferrin(HLF),a pivotal iron-binding glycoprotein belonging to the transferrin family,is the primary immune protein in breast milk.Currently,recombinant HLF expression in microorganisms has attracted widespre...Human lactoferrin(HLF),a pivotal iron-binding glycoprotein belonging to the transferrin family,is the primary immune protein in breast milk.Currently,recombinant HLF expression in microorganisms has attracted widespread attention.Through genome integration and copy number optimization,this study established a Komagataella phaffii cell factory that achieved an HLF titer of 137.6 mg/L.However,HLF accumulated extensively within the cells.Subsequently,several endogenous signal peptides were screened and hybridized with mating factorα,resulting in a 1.56-fold increase in extracellular HLF titers compared to the control.Additionally,protein kinase Ⅱ overexpression facilitated vesicle trafficking,further increasing extracellular HLF to a titer of 304.6 mg/L in shake flasks and 1.7 g/L in a 3-L bioreactor.Notably,the intracellular/extracellular HLF concentration ratio decreased from 1.69:1 to 1.01:1.Further enhancements were made through ion optimization and an exponential methanol-feeding strategy,resulting in an extracellular HLF titer of 3.1 g/L.To the best of our knowledge,this is the first study to report considerably improved extracellular HLF production through optimization of the transport and secretion pathways and provide insights into the expression of other secretary human proteins in K.phaffii.展开更多
AIM: To study the effect of seminal plasma on Chemokine(C-C motif) ligand 20(CCL20) production by epithelial cells and its relationship with lactoferrin.METHODS: HEC-1A cells, a cell line derived from a monostratified...AIM: To study the effect of seminal plasma on Chemokine(C-C motif) ligand 20(CCL20) production by epithelial cells and its relationship with lactoferrin.METHODS: HEC-1A cells, a cell line derived from a monostratified endocervical epithelium, were incubated with samples of seminal plasma(diluted 1:10 in culture medium) recovered from human immunodeficiency virus(HIV) seronegative(HIV-) or HIV seropositive(HIV+) subjects. Recombinant human interleukin 1 beta(IL-1β) was used as positive control, and culture medium only as negative control. The measurement of CCL20 production in the supernatants of HEC-1A cells and of lactoferrin in seminal plasma was determined by enzyme-linked immunosorbent assay techniques. A fractionation of seminal plasma proteins was performed by ion exchange chromatography on a pool of seminal plasma specimens from HIV- subjects. Each fraction was tested for its ability to stimulate the production of CCL20 by HEC-1A cells and for its lactoferrin concentration. The HIV viral load in seminal plasma samples from HIV+ patients was measured using the HIV-Monitor kit(Roche Diagnostic Systems, Branchburg, NJ, United States).RESULTS: The positive control IL-1β was responsible for an increase of 11.36 ± 3.36 times in the production of CCL20. Stimulation of HEC-1A cells was performed in 34 seminal plasma samples(22 from HIV+ subjects and 12 from HIV- subjects). The mean production of CCL20 by HEC-1A in presence of seminal plasma from HIV- and HIV+ subjects was respectively 5.38 ± 0.91 and 7.57 ± 3.26 times higher than that obtained with the untreated cells(P < 0.05 between the two groups). Using the same 34 specimens of seminal plasma, no correlation was observed between the concentration of total proteins in seminal plasma and their ability to stimulate the secretion of CCL20 by HEC-1 cells. In contrast, the ability to produce CCL20 by HEC-1A cells correlated to the concentration of lactoferrin in the seminal plasma samples(r coefficient = 0.56; CI: 0.26-0.76; P < 0.001). After fractionation by ion exchange chromatography, the seminal plasma fractions exhibiting the highest concentrations of lactoferrin were responsible for the greatest stimulation of CCL20 production by HEC-1A cells(r coefficient = 0.89; CI: 0.78-0.95; P <0.0001). CONCLUSION: Lactoferrin present in seminal plasma correlated with an increased production of CCL20 by HEC-1A cells and therefore could facilitate HIV entry through the genital mucosa.展开更多
为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TAL...为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。展开更多
目的:建立快速、简便测定鲜牛奶、转基因牛奶和人乳中乳铁蛋白的方法。方法:在对样品脱脂和去除酪蛋白时,水洗乳脂、酪蛋白以提高乳铁蛋白的回收率。通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate-polyacrylamide gel e...目的:建立快速、简便测定鲜牛奶、转基因牛奶和人乳中乳铁蛋白的方法。方法:在对样品脱脂和去除酪蛋白时,水洗乳脂、酪蛋白以提高乳铁蛋白的回收率。通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分离乳清蛋白,薄层扫描法定量。对电泳和薄层扫描的条件进行优化,电泳使用1.0mm×10齿的试样格、分离胶质量浓度12g/mL、分离电压100V、上样量5μL、染色3h、脱色2h;薄层扫描采取锯齿、双波长、透射的扫描方式,Y步长和摆幅宽分别为0.1mm和8mm。结果:可以分离不同来源乳中的乳铁蛋白、α-乳白蛋白和β-乳球蛋白;乳铁蛋白加标回收率分别为104.53%、108.37%,同板精密度RSD值为3.1003%和1.8151%,在100～2000μg/mL范围内呈线性关系,相关系数为0.9988和0.9990。结论:此方法可以用于3种乳中乳铁蛋白的测定。展开更多
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
基金Supported by Genetically Modified Organisms Breeding Major Projects (2008ZX08007-001)~~
文摘[Objective] The aim was to study whether recombinant Human Lactoferrin has toxic effect on Primary Hepatocyte and Nephrocyte Cell of rat to provide reference for further safety evaluation.[Method] Recombinant Human Lactoferrin and its digested products were taken as tested compound,cow Lactoferrin was used for contrast.Primary Hepatocyte and Nephrocyte Cell of rat were cultured and IC50 values were tested by MTT,and cytotoxic dose-response relationship was tested.[Result]Target toxicity was not found from recombinant Human Lactoferrin on hepatocytes and nephrocytes,in accordance with sub-chronic toxicity test.[Conclusion] This study is of reference value for further safety evaluation of recombinant Human Lactoferrin and safety of evaluation method of GM food.
基金supported by Agricultural Science and Technology Major Project,Basic Research Program of Jiangsu(BK20251622)Jiangsu Funding Program for Excellent Postdoctoral Talent(2025ZB704)the State Key Laboratory of Food Science and Tech-nology,Jiangnan University(SKLF-ZZB-202310).
文摘Human lactoferrin(HLF)belongs to the transferrin family and serves as a crucial component of the innate im-mune system.Traditionally,lactoferrin is primarily extracted from whey.However,this method is constrained by low efficiency and high cost.Microbial cell factories have emerged as an important alternative solution to address this issue.This study aimed to achieve HLF expression by evaluating two host strains of Aspergillus niger,along with suitable promoters and signal peptides.Subsequently,we identified an appropriate glucoamylase(GlaA)fusion fragment to enhance HLF expression and secretion.The knockout of the proteases PepA and PepB reduced HLF degradation after secretion.Subsequently,a multi-gene integration strategy was employed to integrate HLF into three highly expressed loci and two protease loci,resulting in a yield of 54.3 mg/L.The knockout of the gene racA that regulates the polar growth of mycelia increased the branching frequency of A.niger hyphae,leading to a yield of 97.7 mg/L.Finally,we achieved a lactoferrin yield of 1027.1 mg/L through fermentation optimization in a 5-L bioreactor.The strategies adopted in this study provides a basis for the application of HLF in A.niger.
基金supported by the National Natural Science Foundation of China(No.32270096)the National Natural Science Foundation of China(No.32021005).
文摘Human lactoferrin(HLF),a pivotal iron-binding glycoprotein belonging to the transferrin family,is the primary immune protein in breast milk.Currently,recombinant HLF expression in microorganisms has attracted widespread attention.Through genome integration and copy number optimization,this study established a Komagataella phaffii cell factory that achieved an HLF titer of 137.6 mg/L.However,HLF accumulated extensively within the cells.Subsequently,several endogenous signal peptides were screened and hybridized with mating factorα,resulting in a 1.56-fold increase in extracellular HLF titers compared to the control.Additionally,protein kinase Ⅱ overexpression facilitated vesicle trafficking,further increasing extracellular HLF to a titer of 304.6 mg/L in shake flasks and 1.7 g/L in a 3-L bioreactor.Notably,the intracellular/extracellular HLF concentration ratio decreased from 1.69:1 to 1.01:1.Further enhancements were made through ion optimization and an exponential methanol-feeding strategy,resulting in an extracellular HLF titer of 3.1 g/L.To the best of our knowledge,this is the first study to report considerably improved extracellular HLF production through optimization of the transport and secretion pathways and provide insights into the expression of other secretary human proteins in K.phaffii.
基金Supported by The Brazilian Federal Agency CAPES(Coordenao de Aperfeioamento de Pessoal de nível Superior)evaluation of graduate education
文摘AIM: To study the effect of seminal plasma on Chemokine(C-C motif) ligand 20(CCL20) production by epithelial cells and its relationship with lactoferrin.METHODS: HEC-1A cells, a cell line derived from a monostratified endocervical epithelium, were incubated with samples of seminal plasma(diluted 1:10 in culture medium) recovered from human immunodeficiency virus(HIV) seronegative(HIV-) or HIV seropositive(HIV+) subjects. Recombinant human interleukin 1 beta(IL-1β) was used as positive control, and culture medium only as negative control. The measurement of CCL20 production in the supernatants of HEC-1A cells and of lactoferrin in seminal plasma was determined by enzyme-linked immunosorbent assay techniques. A fractionation of seminal plasma proteins was performed by ion exchange chromatography on a pool of seminal plasma specimens from HIV- subjects. Each fraction was tested for its ability to stimulate the production of CCL20 by HEC-1A cells and for its lactoferrin concentration. The HIV viral load in seminal plasma samples from HIV+ patients was measured using the HIV-Monitor kit(Roche Diagnostic Systems, Branchburg, NJ, United States).RESULTS: The positive control IL-1β was responsible for an increase of 11.36 ± 3.36 times in the production of CCL20. Stimulation of HEC-1A cells was performed in 34 seminal plasma samples(22 from HIV+ subjects and 12 from HIV- subjects). The mean production of CCL20 by HEC-1A in presence of seminal plasma from HIV- and HIV+ subjects was respectively 5.38 ± 0.91 and 7.57 ± 3.26 times higher than that obtained with the untreated cells(P < 0.05 between the two groups). Using the same 34 specimens of seminal plasma, no correlation was observed between the concentration of total proteins in seminal plasma and their ability to stimulate the secretion of CCL20 by HEC-1 cells. In contrast, the ability to produce CCL20 by HEC-1A cells correlated to the concentration of lactoferrin in the seminal plasma samples(r coefficient = 0.56; CI: 0.26-0.76; P < 0.001). After fractionation by ion exchange chromatography, the seminal plasma fractions exhibiting the highest concentrations of lactoferrin were responsible for the greatest stimulation of CCL20 production by HEC-1A cells(r coefficient = 0.89; CI: 0.78-0.95; P <0.0001). CONCLUSION: Lactoferrin present in seminal plasma correlated with an increased production of CCL20 by HEC-1A cells and therefore could facilitate HIV entry through the genital mucosa.
文摘为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。
文摘目的:建立快速、简便测定鲜牛奶、转基因牛奶和人乳中乳铁蛋白的方法。方法:在对样品脱脂和去除酪蛋白时,水洗乳脂、酪蛋白以提高乳铁蛋白的回收率。通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分离乳清蛋白,薄层扫描法定量。对电泳和薄层扫描的条件进行优化,电泳使用1.0mm×10齿的试样格、分离胶质量浓度12g/mL、分离电压100V、上样量5μL、染色3h、脱色2h;薄层扫描采取锯齿、双波长、透射的扫描方式,Y步长和摆幅宽分别为0.1mm和8mm。结果:可以分离不同来源乳中的乳铁蛋白、α-乳白蛋白和β-乳球蛋白;乳铁蛋白加标回收率分别为104.53%、108.37%,同板精密度RSD值为3.1003%和1.8151%,在100～2000μg/mL范围内呈线性关系,相关系数为0.9988和0.9990。结论:此方法可以用于3种乳中乳铁蛋白的测定。
