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1.
High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice 总被引:2,自引:1,他引:1
Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells. 相似文献
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Expression of hepatitis C virus non-structural 5A protein in the liver of transgenic mice 总被引:7,自引:0,他引:7
Majumder M Steele R Ghosh AK Zhou XY Thornburg L Ray R Phillips NJ Ray RB 《FEBS letters》2003,555(3):528-532
4.
Lisauskas SF Cunha NB Vianna GR Mendes EA Ramos GL Maranhão AQ Brígido MM Almeida JO Baptista HA Motta FL Pesquero JB Aragão FJ Rech EL 《Biotechnology letters》2008,30(12):2063-2069
Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving
coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed
to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk
(3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the
FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed
in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of
coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the
mammary gland of transgenic mice. 相似文献
5.
Hepatitis B is the most common and serious liver disease, especially in developing countries. Although HBV pathogenesis has been extensively investigated, the proteomic alteration of hepatocytes during HBV chronic infection is still unclear. Using the purified hepatocytes, we compared the protein profiles by 2‐DE and LC‐MS between HBV‐transgenic (Tg) and corresponding background mice. Twenty‐seven altered proteins were identified in hepatocytes from HBV‐Tg mice, among which 13 proteins were involved in mitochondrion metabolism pathway including tricarboxylic acid (TCA) cycle and oxidative response; four proteins (SELENBP, SCP2, RGN and PRDX1) were also dramatically changed in liver samples from HBV‐infected patients. Important genes (gpx, sod, ogg et al.) correlated to oxidative damage were up‐regulated in the liver of HBV‐Tg mice. Reactive oxygen species production was significantly increased while ATP production was decreased in liver mitochondria from HBV‐Tg mice. Moreover, hepatocytes of HBV‐Tg mice were more sensitive to hydrogen peroxide‐induced cell death than that of wild‐type control. Using 2‐D Western blotting analysis, eight hepatocyte proteins were found to react with sera of HBV‐Tg mice but not with that of background mice. Interestingly, two (Etfa and Dmgdh) of the eight reactive proteins were overexpressed in HBV‐Tg mice. We believe this study is the first proteomic and seroproteome analysis of HBV‐infected mammalian hepatocyte and provides insightful links between HBV infection and HBV‐induced liver diseases. 相似文献
6.
Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (= 280 ATU/g wet tissue); however, mammary glands showed a higher activity of 780 ATU/g wet tissue. Transgenic mice showed no evident physical abnormality. The purified rHirudin was further analyzed by amino acid analysis and was found to contain a tyrosine O-sulfate residue that is absent in rHirudin expression either through Escherichia coli or yeast host systems. Experimental results demonstrated that the beta-casein-promoted Hirudin transgene could be successfully expressed in a murine model and may be applicable to large mammals such as livestock for mass production of rHirudin for pharmaceuticals. 相似文献
7.
Valdés R Reyes B Alvarez T García J Montero JA Figueroa A Gómez L Padilla S Geada D Abrahantes MC Dorta L Fernández D Mendoza O Ramirez N Rodriguez M Pujol M Borroto C Brito J 《Biochemical and biophysical research communications》2003,310(3):742-747
This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice. 相似文献
8.
Yuskevich V Khodarovich Y Kagarliskiy G Stremovskiy O Maksimenko O Lukash S Polanovsky O Deyev S 《Biochimie》2011,93(3):628-630
A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (Kd = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals. 相似文献
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Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk 总被引:1,自引:0,他引:1
Baldassarre H Hockley DK Doré M Brochu E Hakier B Zhao X Bordignon V 《Transgenic research》2008,17(1):73-84
The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade,
as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional
systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have
been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be
associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the
mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing
recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range
1–5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing
these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an
apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes
in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount
of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the
blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications.
As a result, this development constitutes another successful example in the application of transgenic animal technology. 相似文献
11.
V. Z. Tarantul V. V. Kucheriavy I. V. Makarova Yu. N. Baranov T. V. Begetova L. E. Andreeva K. G. Gazaryan 《Molecular & general genetics : MGG》1986,203(2):305-311
Summary Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the plasmid rescue technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed. 相似文献
12.
Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora 总被引:4,自引:0,他引:4
Lysozyme is a key antimicrobial component of human milk that has several health-promoting functions including the development
of a healthy intestinal tract. However, levels of lysozyme in the milk of dairy animals are negligible. We have generated
transgenic dairy goats that express human lysozyme (HLZ) in their milk in an attempt to deliver the benefits of human milk
in a continual fashion. To test the feasibility of this transgenic approach to achieve a biological impact at the level of
the intestine, feeding trials were conducted in two animal models. Pasteurized milk from HLZ transgenic animals was fed to
both kid goats (ruminant model) and young pigs (human model), and the numbers of total coliforms and Escherichia coli present in the small intestine were determined. Data from this proof-of-principle study demonstrate that milk from transgenic
animals was capable of modulating the bacterial population of the gut in both animal models. Pigs that consumed pasteurized
milk from HLZ transgenic goats had fewer numbers of coliforms and E. coli in their intestine than did those receiving milk from non-transgenic control animals. The opposite effect was seen in goats.
Milk from these transgenic animals not only represent one of the first transgenic food products with the potential of benefiting
human health, but are also a unique model to study the development and role of intestinal microflora on health, well-being
and resistance to disease. 相似文献
13.
Zhang J Li L Cai Y Xu X Chen J Wu Y Yu H Yu G Liu S Zhang A Chen J Cheng G 《Protein expression and purification》2008,57(2):127-135
This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats. 相似文献
14.
Expression of human serum albumin in the milk of transgenic mice 总被引:7,自引:0,他引:7
Moshe Shani Itamar Barash Margret Nathan George Ricca George H. Searfoss Itzhak Dekel Alexander Faerman David Givol David R. Hurwitz 《Transgenic research》1992,1(5):195-208
We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock
by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used
to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3,
5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression
of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were
used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated
from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with
an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg
ml−1) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated
from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from
1 to 35 μg ml−1. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced
SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene
containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals,
that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate
the relative expression efficiencies of various vector constructs intended for future transgenic evaluation. 相似文献
15.
Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine -lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells. 相似文献
16.
Yaroslav Gursky Robert Bibilashvili Mikchail Minashkin Alex Krasnov Alex Deikin Tatyana Ermolkevich Andrey Popov Lilia Verbovaya Nicolai Rutkevich Alexsander Shevelev Sofia Georgieva Sergey V. Razin Igor Goldman Elena Sadchikova 《Transgenic research》2009,18(5):747-756
Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase
by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk
of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified
ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type
plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification
of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing
the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along
with a protease inhibitor. 相似文献
17.
Ren J Wang L Liu G Zhang W Sheng Z Wang Z Fei J 《Acta biochimica et biophysica Sinica》2008,40(2):111-115
Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgeulc animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation. 相似文献
18.
目的建立系统性表达人载脂蛋白A1(APOA1)基因的转基因小鼠。方法 将人APOA1基因插入系统性表达启动子下游,构建转基因表达载体,通过显微注射法建立人APOA1转基因C57BL/6J小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,Western blot检测基因表达水平,血生化分析检测不同月龄转基因小鼠与同龄野生型小鼠的血脂指标。结果建立了2个不同表达水平的人APOA1基因的转基因小鼠品系;转入的人APOA1基因在血液、肝脏、心脏、肾脏、脾脏、血管组织中均有明显表达;血生化分析结果显示不同月龄转基因小鼠的血浆高密度脂蛋白胆固醇水平高于同龄的野生型小鼠,甘油三酯水平低于同龄野生型小鼠。结论成功建立了系统性表达人APOA1基因的转基因小鼠,为研究高血脂以及高血脂相关的心血管病提供了工具。 相似文献
19.
Gallozzi M Chapuis J Le Provost F Le Dur A Morgenthaler C Peyre C Daniel-Carlier N Pailhoux E Vilotte M Passet B Herzog L Beringue V Costa J Tixador P Tilly G Laude H Vilotte JL 《Transgenic research》2008,17(5):783-791
RNA interference has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. A designed miRNA targeting the prion protein mRNA was built and expressed using the human PRNP promoter. Its efficiency was confirmed in transfected cells and it was used to generate several transgenic mouse lines. Although expressed at low levels, it was found to downregulate the endogenous mouse Prnp gene expression to an extent that appears to be directly related with the transgene expression level and that could reach up to 80% inhibition. This result highlights the potential and limitations of the RNA interference approach when applied to disease resistance. 相似文献
20.
目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。 相似文献