人血清白蛋白基因乳腺表达载体在奶山羊乳腺中的瞬间表达

构建了包括人血清白蛋白 ( h ALB)基因的 c DNA全序列、内含子 1以及山羊β-酪蛋白基因启动子和 5′端上游调控序列在内的乳腺表达载体 pc DNA3.1 -GCALBm,采用直接注射法将其导入哺乳期的奶山羊乳腺组织 ,然后通过放射免疫法测定乳汁中 h ALB的含量。结果显示 ,5头受试奶山羊的乳汁中有明显的 h ALB表达 ,注射后第 2天放免测定值从注射前的 4 .0 mg/L左右分别上升至 62 .2、59.2、36.2、4 2 .3、1 7.5mg/L,并持续表达在 2周以上 ,其中 1头受试羊乳汁中 h ALB的表达量维持在 80 mg/L左右 ,并超过了 1 0 0 d。而在乳腺组织中注射 pc DNA3.1空质粒或生理盐水的 4头对照奶山羊的乳汁中 ,放免测定值无变化。     

hEPO乳腺定位表达载体的构建及其在大鼠和家兔乳腺中的暂时表达

将人红细胞生成素（ｈＥＰＯ）基因组ＤＮＡ（ｇＤＮＡ）与大鼠乳清酸蛋白（ＷＡＰ）基因的上游调控序列融合,分别构建了ＰＷＡＰＥＰＯ－ＷＡＰ３‘ＵＴＲ（ＰＷＥ３’）和ＰＣＭＶ－ＷＡＰ－ＥＰＯ－ＢＨＧｐｏｌｙ（ｐＣＷＥＡ）２个乳腺中位表达载体,表达载体经脂质体包裹后,以显微外科方法,经乳腺导管注入妊娠中后期大鼠和家兔腺内,进行暂时表达。分别大于分娩后４８、７２ｈ,家兔分娩后１－１０ｄ采奶,ＥＬＩＳＡ方法检     

猫血清白蛋白在毕赤酵母中的分泌表达

【目的】在毕赤酵母中构建猫血清白蛋白(feline serum albumin, FSA)表达系统,并探索其最适的基本表达条件,为FSA的生产提供一种新方法。【方法】在GenBank上获取FSA碱基序列,进行密码子优化并合成,将优化的密码子构建到载体pPIC9K上,经PCR和双酶切验证后通过电击转化将重组质粒FSA-pPIC9K转化毕赤酵母GS115,依次经MD平板和G418筛选后进行菌落PCR获得阳性菌株,随机选取阳性菌株经甲醇诱导表达96 h后,取表达上清液进行Western blotting验证。选取表达量较高的菌株分别在不同温度(24、26、28和30℃)、pH(4.0、5.0、6.0、7.0和8.0)和甲醇诱导量(其中1组为每24 h添加0.5%,其余4组为每12 h分别添加0.5%、1.0%、1.5%和2.0%)的条件下诱导表达96 h,取表达上清液进行Western blotting验证。【结果】菌落PCR结果显示,获得2条大小分别约为2.3和2.2 kb的目的基因条带和毕赤酵母AOX1基因扩增条带,诱导表达96 h后取表达上清液进行Western blotting验证,...     

奶牛bTAP蛋白表达载体的构建及其在奶牛乳腺上皮细胞中的表达

奶牛气管抗菌肽是一种具有较强抗菌活性的抗菌小肽。实验从奶牛气管组织中提取总RNA,反转录成c DNA,以此为模板,用奶牛气管抗菌肽特异性PCR引物扩增奶牛气管抗菌肽基因,然后将其连入载体p CMV-C-e GFP中构建奶牛气管抗菌肽的真核表达载体b TAP-GFP,并将此载体转染进奶牛乳腺上皮细胞中,分别运用荧光蛋白观察和细菌记数等方法检测b TAP-GFP蛋白在细胞中的表达及其对大肠杆菌的抗菌活性。试验结果表明,试验成功的建立了奶牛气管抗菌肽的真核表达载体b TAP-GFP,并且此重组载体能在奶牛乳腺上皮细胞中正常表达具有抗大肠杆菌活性的b TAP-GFP蛋白。     

人血清白蛋白DNA片段的扩增及克隆

根据人血清白蛋白(HSA)基因的第八个外显子区DNA序列及相应cDNA序列和KB小鼠血清蛋白DNA序列设计的一对引物P_1:5'-GGATCCACCAACTTACTTATAGGCG-3'和P_2:5'-AGGATCCTACTTACATGCCCAGGAAG-3'.以人白细胞和KB鼠DNA为模板,在50_(μl)PCR反应体系中,经94℃、lmin,58.5℃,35S,72℃、lmin三个循环.94℃、lmin,57.5℃,40S.72℃、lmin32个循环扩增,获得了约为256bp的单一带DNA,KB鼠无专一带.将人HSA DNA扩增的单一带DNA以BamHI-Sst I位点克隆到pUC19中,得到HSA DNA扩增片段的克隆子.     

柔嫩艾美耳球虫Serpin基因真核表达载体的构建及其瞬时表达

为了解柔嫩艾美耳球虫(E.tenella) Serpin基因的生物学特性,本研究采用PCR技术扩增去除信号肽后的Serpin基因,得到1 204 bp的特异性片段,并构建重组表达质粒pVAX1-Serpin,将其瞬时转染HeLa细胞.间接免疫荧光检测表明Serpin基因在HeLa细胞中得到了表达,western blot显示表达蛋白分子量约为47 ku,具有较好的反应原性.该研究结果为E.tenella Serpin核酸疫苗的研究奠定了基础.     

人血清白蛋白（HSA）轩基因小鼠的制备

以绵羊金属硫蛋白基因（ｍＴＭ）为启动子，构建了２种结构的ＨＳＡ质粒：ｐｏＭＴＨＳＡ和ｐｏＭＴＨＳＡＵＤ。其中ｐｏＭＴＨＳＡ是将ＨＳＡｃＤＮＡ直接置于ｏＭＴ启动子的控制下，以ＰＳＰＯＲＴ１为载体构建而成；而ＰＯＭＴＨＳＡＵＤ另融有ＨＳＡ基因５，３侧翼序列（包括内含子１，２，１３，１４）。将这２种结构的质粒用ＮｏｔⅠ线化后，注入昆明种小鼠受精卵的雄原核，再移植到假孕受体鼠的输卵管。共注射５７７枚受精     

人胰岛素基因杆状病毒表达载体的构建及其在Sf9细胞中的表达

对人胰岛素基因真核表达载体（pCMA／mINS）进行Xho I酶切，回收的含人胰岛素基因组基因的1608bp片段与线性化的杆状病载体pFast Bac I进行连接，获得重组载体pFast/mINS。将该重组载体转化DH10Bac感受态细菌，在体内进行重组，并经2次抗性与蓝白斑筛选，得到杆状病毒重组载体Bacmid/mINS。将该载体转染Sf9细胞，获得了重组杆状病毒，经Tricine-SDS-PAGE和Western-blotting检测，重组杆状病毒在Sf9细胞中的表达产物是胰岛素原，而细胞培养上清中未检测到胰岛素和胰岛素原。     

转基因动物乳腺通用表达载体的构建

为进一步研究羊β乳球蛋白（ＢＬＧ）基因在转基因大动物中的应用,利用克隆的羊ＢＬＧ基因５′和３′区的５ｋｂ和４．２ｋｂ构建乳腺表达载体。为有效利用内含子的作用,克隆了羊ＢＬＧ第１和第２内含子,进行了序列分析,并将２个内含子进行了拼接,构建了有效的乳腺表达载体。该载体方便从原核载体中卸出,并加入３个有利于外源基因插入的克隆位点     

人血清白蛋白基因在鹌鹑两种细胞上表达的研究

培养鹌鹑输卵管上皮细胞和鹌鹑胚胎成纤维细胞作为检测平台,将重组后的人血清白蛋白表达载体用脂质体包埋法转染以上两种细胞,通过荧光显微镜观察到报告基因绿色荧光蛋白的表达,结果表明构建的表达载体在成纤维细胞上未见表达;而原代输卵管上皮细胞上有表达。取转染细胞的基因组DNA,设计特异性的检测引物,用PCR筛选法检测了报告基因表达的阳性细胞克隆,SDS-PAGE银染色法确定鹌鹑输卵管上皮细胞分泌蛋白的分子量,结果显示在转染的鹌鹑输卵管上皮细胞培养上清中含有分子量约为68kDa的HSA。表明构建的表达载体是有效的,而且表达构件已经初步整合到阳性鹌鹑输卵管上皮细胞的染色体中。     

绵羊肺炎支原体标准株Y98外膜蛋白MOP30基因真核表达载体的构建及其在烟草中的瞬时表达

以绵羊肺炎支原体（Mycoplasma oumvipneoniae,MO）标准株Y98基因组为模板,设计1对特异引物,PCR扩增得到798bp的P30目的基因（MOP30）,将其定向克隆到pMD19-T Simple载体中进行测序分析。重组质粒进行XbaⅠ/BamHⅠ双酶切并回收目的片段,将目的基因亚克隆入黄色荧光蛋白（yellow fluorescent protein,YFP）表达载体pCAMBIA1300-YFP中构建重组融合表达质粒pCAMBIA1300-MOP30-YFP。双酶切验证后的融合表达质粒转化农杆菌（Agrobacterium）感受态细胞,侵染烟草（tobacco）叶片。通过激光共聚焦成像显微镜（cofocal imagingmicroscope）观察到融合表达的黄色荧光蛋白,Western-blotting试验得到57 000的特异条带,RT-PCR检测到MOP30基因在烟草叶片中转录。MOP30-YFP融合蛋白在烟草中的成功表达,为转基因植物疫苗防治绵羊支原体肺炎奠定了基础。     

山羊痘病毒P32基因重组表达载体的构建及原核表达

以含山羊痘病毒贵州LD株P32基因的质粒pMD18-T-P32/LD为模板,应用PCR方法扩增P32基因,将该基因片段克隆至原核表达载体pET-28a,构建了重组表达载体pET-28a-P32/LD,转化大肠杆菌BL21(DE3)中并诱导表达。SDS-PAGE分析结果显示,有分子量约为35.5 ku的融合蛋白获得表达,与预期结果相符;Western-Blotting证实该表达蛋白具有免疫学活性。P32蛋白的成功表达为山羊痘基因工程疫苗的研制及诊断方法的建立奠定了基础。     

猪IL-15 cDNA真核表达载体的构建、表达及其免疫佐剂效应

参考GenBank发表猪IL-15mRNA序列设计引物,用RT-PCR方法扩增猪IL-15cDNA,并克隆到pMD18-T载体中,通过PCR、酶切和测序验证克隆正确,再亚克隆到真核表达载体pcDNA-3.1（＋）上,得到重组质粒pcD-NA-pIL-15。在脂质体介导下,重组质粒pcDNA-pIL-15转染AD-293细胞。以鼠抗猪IL-15为一抗,用间接免疫荧光分析表明猪IL-15基因均在AD-293细胞中成功进行了瞬时表达。小鼠免疫试验表明,pcDNA-pIL-15作为免疫佐剂,能够有效提高小鼠的脾T细胞增殖,加强pcDNA-ORF2（PCV2）质粒免疫过程中的特异性中和抗体的产生,为进一步研制猪IL-15基因佐剂疫苗及进行临床实验奠定基础。     

Modulation of apical Na+/Pi cotransporter type IIb expression in epithelial cells of goat mammary glands

Recently, the type II sodium-dependent phosphate cotransporter NaPi IIb, which mediates the intestinal phosphate transport, was detected in the apical membranes of caprine mammary gland epithelial cells. Regulatory influences of developmental stages, dietary phosphorus (P) supply and hormones like calcitriol are well described for the intestinal NaPi IIb. Therefore, it was the aim of this study to examine the influence of involution and dietary P restriction on the expression of mammary gland NaPi IIb and of vitamin D receptor (VDR). During involution both, NaPi IIb and VDR, showed an initial increase of expression. This resulted in a delayed response on protein level. Dietary P restriction resulted in a decrease of mRNA expression which was not reflected on protein level. Influenced expression pattern, at least on mRNA level, indicate that mammary gland NaPi IIb is a regulated phosphate transporter which might have an important role especially during involution. Coexpression pattern with VDR provides an indication that calcitriol could be the modulator of these adaptive responses to involution and dietary P restriction. Therefore, a physiological meaning of NaPi IIb in mammary gland epithelia during processes of cell regeneration has to be considered.     

大鼠不同时期乳腺组织及血液中催乳素变化规律

为探讨催乳素在大鼠不同时期乳腺及血液中的变化规律,选择雌性SD大鼠42只,分别为处女鼠、妊娠鼠(6 d、12 d、18 d)、泌乳鼠(6 d、12 d、18 d),每个时期6只.在指定的时间点处死大鼠取乳腺组织及血清.用放免法对催乳素表达的变化进行定量研究,用统计学方法进行处理,观察催乳素的变化规律.结果显示,血清PRL水平以处女期最高,妊娠开始急剧下降,随着妊娠进行又逐渐增加,分娩后随着泌乳进行期渐渐下降.处女期与其余各期差异显著(P＜0.05),整个妊娠期间以妊娠18 d为最高,且妊娠期间差异不显著(P＞0.05),泌乳6 d明显高于泌乳12 d、18 d(P＜0.05),泌乳12 d、18 d明显低于妊娠各期水平(P＜0.05).乳腺PRL变化模式与血清的变化不一致,处女期最高,随妊娠下降,泌乳期相对稳定,变化不大.处女期与其余各期差异显著(P＜0.05),妊娠6 d水平仅明显低于处女期(P＜0.05),而显著高于其余各期(P＜0.05).表明催乳素不仅参与了乳腺细胞的发育,而且在维持和启动泌乳方面也起到重要的作用.     

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT‐MECs)

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post‐parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT‐MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF‐7 cells. hT‐MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT‐MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta‐casein protein was synthesized and secreted. Beta‐casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT‐MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT‐MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT‐MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.     

Haemoglobin in normal and neoplastic canine mammary glands

Four types of globins for oxygen transport are known in vertebrates, and the haemoglobin is responsible for carrying oxygen in blood. In this study, we found that haemoglobin was also expressed in canine mammary glands. Samples were taken from 26 malignant mammary tumors, 16 normal mammary glands and 10 other normal tissues. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and mass spectrometry were used to investigate haemoglobin in mammary tissues. The results indicated that normal canine mammary glands expressed high levels of haemoglobin protein as shown by Coomassie blue staining. The identity of haemoglobin was confirmed by immunoblotting and mass spectrometry, and the mass spectrometry data revealed that both alpha-haemoglobin and beta-haemoglobin were expressed. Relative to normal mammary glands, the levels of haemoglobin expression in mammary tumors were lower. Our results also indicated that the haemoglobin was endogenously produced in mammary gland tissues and was not derived from the erythroid cells.     

真核表达载体pIRES2-EGFP-V13KL的构建及在COS-7细胞和小鼠胚胎中的表达

构建了真核表达载体pIRES2-EGFP-V13KL,瞬时转染COS-7细胞及对小鼠受精卵进行原核注射,旨在通过抗菌肽V13KL在COS-7细胞、小鼠胚胎中的表达情况评价其对细胞生理状态、胚胎发育方面可能产生的影响。结果显示,V13KL可以与EGFP在COS-7细胞中高效共表达,转染效率为75.8%,且原核注射后约10%的胚胎发育成带有绿色荧光的囊胚。