共查询到20条相似文献,搜索用时 31 毫秒
1.
Han ZS Li QW Zhang ZY Yu YS Xiao B Wu SY Jiang ZL Zhao HW Zhao R Li J 《Journal of microbiology and biotechnology》2008,18(1):153-159
limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3 mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest. 相似文献
2.
Expression of recombinant pharmaceutical proteins in the mammalian mammary gland is of great interest for the medical industry. This study was designed to express recombinant human antithrombin (rhAT) in the mammary gland of rabbits by adenovirus vectors infection. Replication-defective adenovirus encoding human antithrombin complementary DNA (cDNA) was constructed and directly infused into the mammary gland of rabbits via the teat canal. The milk serum was collected from the infected mammary gland 48?h post-infection and subjected to Western blot analysis, Enzyme-linked immunosorbent assay (ELISA), and antithrombotic activity assay. In this way, the target protein was verified, and a high expression level of rhAT up to 4.8?g/L was obtained, and antithrombotic activity of the rhAT was not different than that of a standard human antithrombin protein (p?>?0.05). Compared to previous attempts to produce human antithrombin in the mammary gland of transgenic animals or fractionation the plasma of blood donors, the method for rhAT expression we established would reduce production cost and further increase production efficacy. 相似文献
3.
The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest. 相似文献
4.
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
5.
Infection efficiency is the key issue for gene delivery using adenovirus vector and usually unsatisfactory. In this study, recombinant adenoviruses encoding recombinant human EPO were prepared using the Adeasy system, and injected into the mammary gland of goats via the teat canal. BAPTA was used to treat the mammary gland to facilitate adenoviruses infection compared with EGTA. Milk serum was collected from the infected mammary gland and characterized by ELISAs and Western blotting. Expression level of rhEPO from the group treated by BAPTA was higher than that treated by EGTA. 相似文献
6.
Rodríguez ES Pose AG Moltó MP Espinoza AS Zamora PA Pedroso MS 《Biotechnology journal》2012,7(8):1049-1053
The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors. 相似文献
7.
Cheng Y An LY Yuan YG Wang Y Du FL Yu BL Zhang ZH Huang YZ Yang TJ 《Molecular reproduction and development》2012,79(8):573-585
It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland‐specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β‐casein, bovine αs1‐casein, or goat β‐lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β‐globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme‐linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17–8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7–40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland‐specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary‐specific expression of recombinant hLF in a transgenic mouse model.Mol. Reprod. Dev. 79: 573‐585, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
8.
9.
K Christensen M O Nielsen N Jarl?v 《Comparative biochemistry and physiology. A, Comparative physiology》1989,93(2):477-481
1. Prostacyclin production in mammary gland of two lactating goats measured as the excretion in milk of 6-ketoprostaglandin F1 alpha (6-KPGF1 alpha) was followed for 16 days before, during and after exogenous administration of recombinant bovine growth hormone (GH). 2. 6-KPGF1 alpha was detected in all milk samples in concentrations ranging from 32-99 pg/ml milk independently of the time of sampling. 3. GH-treatment significantly increased milk yield, the concentration and excretion of 6-KPGF1 alpha in milk. 4. The concentration of milk 6-KPGF1 alpha was positively correlated with milk yield in the high (R2 = 0.35), but not in the low yielding goat (R2 = 0.003). 5. The possible role of prostacyclin as a local vasodilator in the mammary gland of goats is discussed. 相似文献
10.
Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland 总被引:1,自引:1,他引:0
Baldassarre H Hockley DK Olaniyan B Brochu E Zhao X Mustafa A Bordignon V 《Transgenic research》2008,17(5):863-872
The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats. 相似文献
11.
Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression
vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin
(hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk.
Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in
which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb
genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding
five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our
strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic
animals. 相似文献
12.
Zakharova ES Pryzhkova MV Kibardin AV Ermolkevich TG Kadulin SG Gnuchev NV Kiselev SL 《Genetika》2005,41(3):299-306
13.
以经过转染的乳腺上皮细胞生产克隆羊 总被引:1,自引:1,他引:1
为研究转基因乳腺上皮细胞发育的全能性,利用电转染方法将人乳铁蛋白(hLF)乳腺特异性表达载体电转染山羊乳腺上皮细胞,经G418和PCR筛选获得阳性克隆细胞株,经催乳素诱导的细胞株上清液用Western blotting方法检测hLF的表达。以转基因与上清液中表达hLF均为阳性的细胞为核供体细胞,进行山羊体细胞核移植。结果为:16株细胞表达重组hLF,分子质量为75 kD;将144枚重构胚移入16只同步发情的山羊输卵管中,在移植后的30 d、60 d和90 d的妊娠率分别为87.5%、81.3%和62.5%;最终3只受体妊娠足月,产下3只克隆羊,克隆效率为2.1%,PCR-RFLP分析表明克隆羊均来自供体羊细胞,但没有整合外源基因。结果表明,hLF转基因乳腺上皮细胞能分泌hLF;乳腺上皮细胞经转染、筛选和长期培养的条件下,能保持发育的全能性。 相似文献
14.
表达人尿激酶原突变体的重组山羊乳腺特异性表达慢病毒载体的构建 总被引:1,自引:0,他引:1
目的:构建山羊乳腺特异性表达尿激酶原突变体的重组慢病毒载体,证明其表达的有效性。方法:将劳氏肉瘤病毒增强子/启动子、复制缺陷型人免疫缺陷病毒(HIV-1)的5′端长重复序列(LTR)、HIV-1ψ包装信号、HIVRev反应元件、山羊β-酪蛋白调控序列、尿激酶原M13cDNA、AU3/3′LTR、牛生长激素(BGH)基因poly(A)依次连接,构建乳腺特异性表达的慢病毒载体,通过体外转染人乳腺癌细胞系MCF-7、中国仓鼠卵巢细胞及泌乳山羊乳腺注射证明其表达有效性。结果:酶切鉴定证实山羊乳腺特异性表达载体构建正确;将该载体转染细胞,采用溶圈法和Western印迹检测证实了其表达的有效性;慢病毒载体注射到泌乳山羊的乳腺,在乳汁中也检测到了尿激酶原的表达。结论:为在转基因动物乳腺中表达尿激酶原突变体奠定了基础。 相似文献
15.
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
16.
17.
Thomassen EA van Veen HA van Berkel PH Nuijens JH Abrahams JP 《Transgenic research》2005,14(4):397-405
Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 Å. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 Å for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.Ellen A. J. Thomassen, Harrie A. van Veen - These authors have contributed equally to this paper.The PDB-code of recombinant human lactoferrin is 2BJJ 相似文献
18.
人血小板生成素(thrombopoietin,TPO)基因组包括6个外显子和5个内含子,内含子在其表达过程中可能扮演着重要作用.为了研究人TPO基因组中不同内含子对TPO表达的影响,构建可在转基因动物乳腺中高水平表达人TPO的乳腺特异性表达质粒.本研究以65 kb的山羊β-casein启动子为调控元件,构建了包括人TPO cDNA(pTPOA)、TPO intronⅠ-TPO cDNA (pTPOB)、ΔTPO intronⅠ-TPO cDNA (pTPOC)、TPO intronⅤ-TPO cDNA (pTPOD)和TPO gDNA (pTPOE)等5种TPO乳腺特异性表达质粒,并在人乳腺细胞HC-11细胞上进行了瞬间表达研究.在转染48 h后,通过双抗体夹心的ELISA方法定量分析上述质粒在HC-11细胞上的表达水平.结果表明,含有内含子Ⅴ的 pTPOD的表达量最高,而含有整个基因组TPO的pTPOE表达水平最低.为了进一步证实pTPOD可在乳腺细胞中高水平表达,将pTPOD经脂质体包埋后注射到泌乳期山羊的乳腺中.结果显示,在山羊乳汁中可连续14 d检测到人TPO的表达.上述实验证实,人TPO基因组中的内含子V可显著提高TPO在HC-11细胞内的表达水平,并提示内含子Ⅴ中可能含有特殊的调控序列. 相似文献
19.
构建携带人尿激酶原突变体cDNA的重组腺病毒,通过该腺病毒介导实现外源基因在山羊乳腺中表达。通过大肠杆菌内同源重组将人尿激酶原突变体cDNA插入到腺病毒载体中,经过293细胞包装获得重组腺病毒,直接注射到泌乳山羊乳腺,收集感染后1~4天的乳汁,利用纤维蛋白溶圈试验、Western blot、ELISA检测乳清中尿激酶原突变体的表达。结果显示病毒注射后1~4天乳清中均可检测到尿激酶原的表达,其表达量可达0.41mg/ml。该方法可以实现重组蛋白在山羊乳腺的短期表达,可能是大规模生产临床医疗蛋白的一条经济有效的途径。 相似文献
20.
Hak-Jae Chung Hyun-Jung Park Sun-Young Baek Jin-Ki Park Won-Young Lee Kyung-Woon Kim 《Animal biotechnology》2013,24(4):317-322
AbstractTissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production. 相似文献