共查询到20条相似文献,搜索用时 96 毫秒
1.
乳腺生物反应器制备中表达载体的发展 总被引:6,自引:1,他引:5
腺生物反应器拥有巨大的开发价值,但开发成功的例子却寥寥可数,主要的原因是外源基因的表达量较低,达不到开发生产的要求。提高外源基因表达量的关键在于乳腺生物反应器表达载体的构建。近年来,这方面的新思路、新方法不断出现,本文对此进行了综述。 相似文献
2.
3.
4.
乳腺生物反应器是指将外源基因导入动物基因组并在动物乳腺中特异性表达,利用动物乳腺合成、分泌蛋白的功能,在其乳汁中获得外源蛋白的技术。乳腺生物反应器凭借其高表达、低成本以及合成蛋白质的结构接近天然蛋白质等优势而被视为药用和营养蛋白生产的一次技术革新,然而由于外源基因随机整合以及重组蛋白表达不稳定等问题极大地限制了其应用。本文结合乳腺生物反应器的发展现状,从利用基因编辑技术、筛选合适的外源基因整合位点以及改进外源基因调控序列3个方面对乳腺生物反应器优化策略进行了综述,以期为提高乳腺生物反应器生产重组蛋白的表达提供理论借鉴。 相似文献
5.
抗凝血酶Ⅲ(AT Ⅲ)是人体内最重要的抗凝血物质,利用转基因技术生产重组人抗凝血酶Ⅲ(rhAT Ⅲ)受到了越来越多的关注,如何有效地将rhAT Ⅲ从转基因羊奶中分离纯化出来是实现其产业化的关键所在。本研究建立了基于等电点沉淀和肝素亲和层析的纯化工艺,实现了rhAT Ⅲ的高效、快速纯化。先用等电点沉淀法,快速去除了占总蛋白50%左右的酪蛋白;然后用肝素亲和层析纯化rhAT Ⅲ,并系统考察了pH值和温度对rhAT Ⅲ稳定性的影响,以及pH值和操作条件(洗脱梯度、流速和上样量)对rhAT Ⅲ分离效果的影响。在优化的条件下,rhAT Ⅲ纯度达到99%以上,蛋白收率达到90%,活性收率约为50%。所建立的纯化工艺简单、快速,rhAT Ⅲ收率高,为今后工艺放大提供了重要的依据和参考。 相似文献
6.
7.
8.
应用转基因技术生产乳腺生物反应器可以获得高效、安全、足量的人类重组蛋白、药用蛋白及其目的蛋白.本文概述了制备转基因动物过程中目的基因选择、载体构建等技术环节的研究现状;通过叙述乳腺生物反应器的原理和应用现状来分析乳腺生物反应器的优势及特点,为乳腺生物反应器的发展和应用奠定理论基础. 相似文献
9.
体细胞基因打靶制备动物乳腺生物反应器的策略与应用 总被引:8,自引:2,他引:8
在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。 相似文献
10.
11.
12.
13.
Lin FY Chen HX Cheng X Xiao Y Deng JX Huang PT 《Acta biochimica et biophysica Sinica》2004,36(7):450-456
The concept of using animal mammary glands asbioreactors to produce recombinant pharmaceuticalproteins has been widely accepted for great potentialcommercial interests [1]. Up to now, the main method tomake transgenic animals is microinjection [2,3]. Lowlevel and unpredictability of the foreign gene expressionwere found among transgenic lines. The major reason isthat the microinjected foreign gene is integrated into thegenome randomly as a stretch of multiple copies, and thesurrounding chromat… 相似文献
14.
15.
Mammary gland polysomes are difficult to isolate from the lactating rat using methods developed for other species and tissues, most likely due to high calcium-stimulated ribonuclease activity in that tissue. A new method, utilizing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) to bind calcium, yields highly aggregated polysomes from lactating rat mammary gland. Fresh mammary tissue is pulverized under liquid nitrogen. Free and membrane-bound polysomes are isolated by differential centrifugation in solutions containing 100 mM KCl, 100 mM MgCl2, 75 mM EGTA, 500 micrograms/ml heparin and 50 mM Tris buffer, pH 8.2 at 5 degrees C. Bound polysomes are released from the endoplasmic reticulum using Triton X-100 and deoxycholate. Polysome profiles are obtained on linear sucrose gradients and scanned at 254 nm. The method gives quantitative recovery of homogenate total RNA. To demonstrate that the method can be used to study nutritional effects on mammary gland polysome aggregation, lactating rats were fasted 22-66 h and then refed a stock diet for 71-95 h. Refeeding increased the percentage of polysomes (trimers or larger) in the bound fraction from 84 +/- 1 to 93 +/- 1% (P less than 0.001) and in the free fraction from 42 +/- 2 to 55 +/- 3% (P less than 0.001). 相似文献
16.
乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定 总被引:1,自引:0,他引:1
以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。 相似文献
17.
Zinovieva Natascha Lassnig Caroline Schams Dieter Besenfelder Urban Wolf Eckhard Müller Sigrid Frenyo Laszlo Seregi Janos Müller Mathias Brem Gottfried 《Transgenic research》1998,7(6):437-447
One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 ± 12g/ml (ranging from 223 ± 61 to 484 ± 39 g/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 ± 41 g/ml (ranging from 360 ± 15 to 678 ± 80 g/ml). Homozygous huIGF-1 females clearly showed a significantly increased production performance of the recombinant protein. 相似文献
18.
A total of 28941 ESTs were sequenced from five 5′-directed non-normalized cDNA libraries, which were assembled into 2212 contigs
and 5642 singlets using CAP3. These sequences were annotated and clustered into 6857 unique genes, 2072 of which having no
functional annotations were considered as novel genes. These genes were further classified into Gene Ontology categories.
By comparing the expression profiles, we identified some breed-and developmental-stage-specific gene groups. These genes may
be relative to reproductive performance or play important roles in milk synthesis, secretion and mammary involution. The unknown
EST sequences and expression profiles at different developmental stages and breeds are very important resources for further
research. 相似文献
19.
Zhixi Su Xinjiao Dong Bing Zhang Yanwu Zeng Yan Fu Jun Yu Songnian Hu 《中国科学:生命科学英文版》2006,49(1):26-36
The mammary gland provides an excellent system to study questions pertaining to organogenesis, cell differentiation and oncogenesis. Intensive efforts have been made to understand the development of the mammary gland, particularly in terms of lactogenesis… 相似文献
20.
To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study. Through the experiment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods. The results are as follows: in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7; progesterone did not affect the expression of FGF7; prolactin up-regulated the expression of FGF7 significantly in pregnancy and lactation. 17 beta-estradiol increased the expression of FGF10; progesterone and prolactin reduced the expression of FGF10 significantly in virgin; prolactin significantly increased the expression of FGF10 in pregnancy. When 17 beta-estradiol in the body was in relatively high proportion, it would lower the expression of KGFR; while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR. Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR. Prolactin increased the expression of KGFR significantly in pregnancy and lactation. 相似文献