山羊β-酪蛋白基因启动子指导的转基因小鼠乳汁高效表达人凝血因子IX

为探讨应用转基因动物乳腺生物反应器高效表达人凝血因子IX(human clotting factor IX,hFIX)的可行性,构建了包括山羊β-酪蛋白基因启动子和外显子l、内含子1、外显子2共约6.7kb片段以及hFIX全长cDNA序列和部分经改造的内含子l的乳腺表达载体,通过转基因技术获得12个原代转基因小鼠(9♀,3♂),整合率为11.2%.经EuSA和western b1ot鉴定8只转基因母鼠乳汁中有hFIX的表达并拥有很高的凝血活性,其中一只的表达量高达52.9mg/L,其凝血活性亦高达279.2%.FISH实验表明不同的转基因小鼠外源基因整合于小鼠的不同染色体上.结果证明所构建的山羊β-酪蛋白基因启动子乳腺表达载体能有效指导hFIX基因在小鼠乳腺中高效表达,并能保持hFIX的生物活性.     

人凝血IX因子基因乳腺组织特异性表达载体的构建及其在奶山羊乳腺中的分泌性表达

血友病B(Hernophilia B)是由于人凝血IX因子(hFIX)缺乏所致的一种严重出血性疾病。常规治疗方法是通过输血或输入人浓缩凝血IX园子来实现的,因此患者很容易被肝炎病毒甚至爱滋病毒感染。近年来,人们期望通过转基因动物乳腺表达并从乳汁中分离生产hFIX蛋白,以解除血友病患者的痛苦。以转基因动物的乳腺组织即乳腺生物反应器生产外源蛋白具有原核生物,酵母以及离俸真核细胞生产系统所不具备的优点：生产的外源蛋白经过体内加工和修饰,特别是真核蛋白的转译后加工(如糖基化和羧基化)．其生化特性、生物学活性与天然蛋白完全相同^[1];另外从乳汁中生产外源蛋白不需要复杂的工厂设备。因此开展人凝血IX因子乳腺生物反应器的研究具有重要的经济意义和临床应用价值。转基因动物乳腺生物反应器的关键在于外源基因乳腺组织特异性表达载体的构建。我们利用小鼠MAR元件(Matrix attachment regions)．牛的β-casein基因调控顺序,hFIX mini-gene和hFIX cDNA分别建成两种乳腺组织特异性表达载体pMCIXm和pMCIX,经SA脂质体包埋载体DNA后直接转染奶山 羊乳腺小叶,hFIX蛋白在奶山革乳汁中获得瞬间分泌性表达,最高表达量为13.7ng/ml乳汁。其中含hFIX mini-gene的表达载体在奶山羊乳汁中的表达量高于含hFIX cDNA的表达载体．表明hFIX的in-tronl能够提高该基因在活体奶山羊乳腺中的表达。A7单抗和柠檬酸钡吸附检测表明乳汁中的hFIX 蛋白羧基化和活性比率都在90％以上。以上结果肯定了载体构建的正确性．不但为研制hFIX蛋白乳腺生物反应器提供了有效的表达载体,同时也表明利用阳离子SA脂质体包埋质粒DNA直接转染活体物乳腺可以作为验证乳腺表达载体构建正确性的快速检测手段。     

铜绿假单胞菌肺部感染小鼠模型中L-17A的表达研究

摘要：目的 了解铜绿假单胞菌肺部感染小鼠模型中L-17A（白介素-17）的表达,指导铜绿假单胞菌肺部感染治疗。方法 择取60只SPF级昆明白雌性小鼠为研究对象,应用随机平行对照法将其分成两组,对照组自小鼠气管内注入相同量的生理盐水,实验组自小鼠气管内注入铜绿假单胞菌悬液0.03 mL,造模后处死两组小鼠,将其肺部组织制制作病理切片,行HE染色,并应用免疫组化法检测肺组织IL-17A表达量变化情况。结果 于3 h、9 h和24 h后,实验组肺组织中IL-17A灰度（182.2±4.7,184.0±9.0,182.8±9.9）明显优于对照组（170.2±4.3,161.8±6.5,147.4±7.3）,且9 h、24 h后血清中IL-17A浓度（132.6±5.5,149.8±5.7）pg/mL显著高于对照组（124.2±2.8,123.2±7.9）pg/mL,差异具有统计学意义（P＜0.05）。结论 IL-17A经由炎症细胞募集介入铜绿假单胞菌肺部感染炎症反应,并参与了病原菌清除过程,临床上应引起足够重视。     

利用烟草表达人凝血IX因子 (hFIX)的研究

血友病B是凝血IX因子（hFIX）缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,本研究构建含人凝血IX因子基因（hFIX,2.8kb）植物双元表达载体p35s-2300：：gus：：noster,用农杆菌介导法转化烟草 "百日红",通过PCR和Southern blot分析证实获得4株独立转基因植株, hFIX在转基因烟草基因组中的拷贝数为1-4个;RT-PCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5~8.8ng/g·FW,并具有免疫活性。本研究为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。     

人促红细胞生成素在转基因小鼠乳汁中表达

将山羊的β乳球蛋白启动子连接人促红细胞生成素基因,构建转基因表达载体。通过显微注射的方法转基因小鼠。经PCR斑点杂交和Southern杂交检测验证,获得4只转基因阳性小鼠,其中3只母鼠哺乳期收集乳汁,经EpoELISA检测为阳性。对4组转基因阳性小鼠的部分子代母鼠,经PCR和Southern杂交分析,又鉴定出6只获得遗传的阳性G1代母鼠以Dot-ELISA的方法检测,其中两只G1代母鼠乳汁中的Epo含量为0.5μg/mL。实验证实,867bp的β乳球蛋白启动子可以指导人促红细胞生成素在小鼠乳汁中表达。     

利用烟草表达人凝血Ⅸ因子(hFIX)的研究

血友病B是凝血Ⅸ因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血Ⅸ因子对血友病B治疗具有重要意义.植物系统表达外源蛋白在生产成本和安全性方面具有优势.为此,构建含人凝血Ⅸ因子基因(hFIX,2.8kb)植物双元表达载体p35s-2300∷gus∷noster,用农杆菌介导法转化烟草"百日红",通过PCR和Southern blot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RT-PCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性.为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考.     

利用烟草表达人凝血IX因子(hFIX)的研究

血友病B是凝血IX因子(hFIX)缺乏所导致的一种出血性疾病,通过输血和hFIX浓缩剂进行治疗疗效显著,但存在治疗费用高和安全隐患,因而获得安全、廉价的人凝血IX因子对血友病B治疗具有重要意义。植物系统表达外源蛋白在生产成本和安全性方面具有优势。为此,构建含人凝血IX因子基因(hFIX,2.8kb)植物双元表达载体p35s2300∷gus∷noster,用农杆菌介导法转化烟草“百日红”,通过PCR和Southernblot分析证实获得4株独立转基因植株,hFIX在转基因烟草基因组中的拷贝数为1～4个;RTPCR和ELISA检测结果表明,hFIX在转录和翻译水平已成功表达,hFIX在转基因烟草叶片中的表达量为2.5～8.8ng/g·FW,并具有免疫活性。为利用植物系统表达hFIX的后续研究作了必要准备,也为利用植物系统表达其他药用蛋白研究提供了一些理论和实验参考。     

ＢＬＧ—ＬＡtPA和鼠ＷＡＰ共注射提高ＬＡtPA在转基因小鼠乳腺中的表达水平

为了提高长效组织纤溶酶原活剂（ＬＡtPA）在转基因小鼠乳腺中的表达水平,将受控于羊β－乳球蛋白（ＢＬＧ）的ＬＡtPA表达载体ＢＬＧ－ＬＡtPA与小鼠乳清酸蛋白（ＷＡＰ）基因片段进行共注射,采用此方法建立的转基因小鼠,经ＰＣＲ筛选和Ｓouthern印迹鉴定,获得３只ＬＡtPA和ＷＡＰ共整合阳性鼠,并在阳性母鼠乳汁中检测到有溶纤活性的ＬＡtPA表达,表达水平达到１０μg/mL。     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白（mWAP)基因5‘端调控区和牛α-sl-酪蛋白基因3‘端调控区作为调节元件构建了用于表达人促血小板生盛开纱的乳腺组织特异性表达载体pWAPTPO(Fig.l)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只,经PCR检测,有6只为转基因阳性（Fig.2)。G0代小鼠中转基因整合率为37.5%(6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8μg/mL以上（Tablel)。这些结果表明我们已建立了乳腺表达hTPO的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

牛BLG基因5＇调控成分的克隆和制备乳腺生物反应器研究

用PCR法克隆了牛β-乳球蛋白(BLG)基因的 1个片段,它包括 650 bp的 5’侧翼序列,第一二外显子和第一内含子.以该片段中的 650 bP的侧翼序列及转录起始位点下游 11 bP的非编码区(共 661 bP)作为调控序列,与人生长激素(hGH)基因构建成了表达载体,在培养的原代山羊乳腺上皮细胞中进行暂时表达,结果在激素诱导下, hGH基因能够表达,并且表达产物的绝大部分分泌到培养基中.将上述表达载体中的BLG/hGH融合基因部分经显微注射法制得5只转基因阳性小鼠,其中1只小鼠乳清中hGH的含量为420 μg/mL而血清中仅为0.051μg/mL,说明本实验克隆的牛BLG基因5’调控序列可以控制目的基因在转基因动物中表达,基本上具有乳腺特异性,而且产物能分泌至乳汁中.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector. Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission.     

Heterochromatin Protein 1 Deleted Chromo Domain Decreases Gene Silencing of Transgene in Mouse

The heterochromatin protein 1 (HP1) regulates epigenetic gene silencing by promoting and maintaining chromatin condensation. To decrease gene silencing, the chromo domain (CD) in the M31 (the main HP1 in mouse) was deleted by site-directed mutagenesis. Vector pcDNA3.1(+)/M31-DeltaCD, in which the M31-DeltaCD is driven by the CMV promoter, and vector pcDNA3.1(+)/P1A3-M31-DeltaCD, in which the M31-DeltaCD is driven by a goat ss-casein promoter were constructed. The former vector was transfected into a murine fibroblast cell line, which can express enhanced green fluorescent protein (EGFP). EGFP expression, which was determined by flow cytometric analysis, increased approximately 80% in the transfected cells. After injection of the latter vector into transgenic mouse mammary glands, which can express human clotting factor IX (hFIX), the hFIX expression level in the mouse milk increased approximately 40-60% and hFIX in one mouse milk was maintained at a high concentration for over 10 days.     

Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor IX (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is evaluated. The muscle creatine kinase enhancer (MCK) and β-actin promoter (βA) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high titer (2.5 × 10¹¹ vector genomes/mL) of rAAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-mediated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

牛β-乳球蛋白基因调控序列指导组织型纤溶酶原激活剂在小鼠乳腺中的表达

用全长8.4kb的牛β－乳球蛋白基因（BLG）作为调控序列,用1.6kb的鸡溶菌酶MAR序列作为对抗转基因中位点效应的工龄,构建了组织型纤溶酶原激活剂（tPA）乳腺表达载体。对2300枚卵进行显微注射,以PCR和Southern-Blot检测,在170只出生小鼠中获得9只整合有牛BLG－tPA融合基因的转基因小鼠,并在转基因小鼠乳汗中检测到tPA r itd ntg ,tPA的表达水平最高达到12μg/mL。整合的小鼠基因组中的牛BLG－tPA融合基因能稳定地遗传给子代。     

Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

Transgenic Mice Can Express Mutant Human Coagulation Factor IX with Higher Level of Clotting Activity

To improve the available values of transgenic animals, we produced a mutant human coagulation factor IX minigene (including cDNA and intron I) with arginine at 338 changed to alanine (R338A-hFIX) by using a direct mutation technique. The R338A-hFIX minigene was then cloned into a plasmid carrying the goat β-casein promoter to get a mammary gland-specific expression vector. The clotting activity in the supernatant of the transfected HC-11 cells increased to approximately three times more than that of wild-type hFIX. Nine transgenic mice (three females and six males) were produced, and the copy number of the foreign gene was very different, ranging from 1 to 43 in different lines. ELISA, Western blot, and clotting assay experiments showed that the transgenic mice could express R338A-hFIX, showing higher average levels of clotting activity than wild-type hFIX in the milk (103.76% vs. 49.95%). The highest concentration and clotting activity of hFIX reached 26 μg/mL and 1287% in one founder (F₀-7), which was over 10 times higher than that in human plasma. Furthermore, RT-PCR, APTT assay, and histological analysis indicated that hFIX was expressed specifically in the mammary gland without affecting the intrinsic coagulation pathway and physiologic performance of the local tissue.     

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100?% efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ??-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.     

A comparative study of hFIX expression mediated by rAAV8 and rAAV1 administrated intramuscularly

BACKGROUND: Recombinant AAV serotype 8 (rAAV8) vector is relatively new for gene therapy. In this study, the hFIX expression mediated by rAAV8 injected intramuscularly was compared with that by rAAV1. METHODS: rAAV8-hFIX or rAAV1-hFIX viruses were injected intramuscularly into two hind limbs of mice at doses of 5x10(10) gc and 2.5x10(12) gc (genome copy). The hFIX expression in the mouse plasma was detected by ELISA, APTT and Western blotting. The virus distribution was analyzed by immunohistochemical assay. RESULTS: When the mice were infected with 5x10(10) gc virus, high levels of hFIX in the plasma of five rAAV8-hFIX virus-infected mice were detected 2 weeks after injection. A hFIX peak above 5000 ng/mL appeared between 2 and 6 weeks after injection. Relatively low levels of hFIX were detected in the plasma of rAAV1-hFIX virus-infected mice 2 weeks after injection. An hFIX peak above 3000 ng/mL appeared between 4 and 10 weeks after injection. However, much lower levels of hFIX were detected in mice infected with higher dose of rAAV8 virus. The hFIX in the mouse plasma was active biologically. The viruses were distributed mainly in the muscles of hind limbs. DISCUSSION: Gene expression mediated by rAAV8 was sooner and stronger than that by rAAV1 after intramuscular administration. Inhibition might have been triggered markedly by rAAV8 at high doses.     

Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.