基于四环素调控系统构建白蛋白启动子调控大鼠uPA转基因表达的慢病毒载体

基于四环素调控系统构建白蛋白(albumin,Alb)启动子调控大鼠uPA(urokinase-type plasminogen activator,ruPA)转基因肝特异性过表达的慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG)。以CTG0875-2-11质粒为模板,PCR扩增大鼠的uPA(ruPA)基因,3’端添加Flag标签,In-Fusion克隆至pLVX-Alb-TetOne-TRE-T2A-CopGFP(pLATTTG)质粒中,得到慢病毒载体pLVX-Alb-TetOne-TRE-ruPA-T2A-CopGFP(pLATTRUTG),所构建质粒经测序和酶切鉴定。将pLATTRUTG瞬时转染293T细胞,转染后24 h倒置荧光显微镜检测CopGFP表达;接着向6孔细胞培养板内加入强力霉素(Doxycycline,Dox),48 h后在倒置荧光显微镜下观察CopGFP表达(包括未加Dox的孔),随后收集细胞以提取总RNA和总蛋白,用于RT-qPCR检测ruPA及报告基因表达和Western blot检测标签蛋白Flag表达。酶切和测序确证我们成功构建了慢病毒载体pLATTRUTG;瞬转293T细胞后,24 h倒置荧光显微镜下可见零星细胞(约占0.1%)发弱的绿色荧光,加Dox 48 h后所有细胞展现强的绿色荧光,而不加Dox的孔内仍然只见到零星细胞(约占0.1%)发弱的绿色荧光。RT-qPCR和Western blot检测结果显示,与不加Dox的细胞相比,加Dox的细胞中ruPA、报告基因CopGFP和Flag表达水平显著升高。结果提示,成功基于四环素调控系统构建Alb启动子调控大鼠uPA转基因表达的慢病毒载体pLATTRUTG,为相关后续实验奠定了基础。

固醇携带蛋白2腺病毒载体的构建与鉴定

目的:构建携带白蛋白启动子SCP2基因腺病毒载体,研究其与胆固醇结石形成的关系。方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2基因过表达时,CYP7a1基因表达下调,(t=3.97,P<0.05)HMGCR基因基因表达上调,(t=3.23,P<0.05)。结论:当SCP2基因过表达时引起HMGCR、CYP7a1基因转录水平的变化,提示SCP2基因可能通过影响胆固醇和胆汁酸的代谢而参与胆固醇结石的形成。

肝特异性表达HCV5′NCR调控荧光素酶质粒的构建及表达

小鼠白蛋白是肝组织特异性表达的蛋白 ,这种特异性是由白蛋白启动子所介导的 .以2 2 35A- 1质粒为模板 ,通过 PCR扩增获得小鼠白蛋白启动子 /增强子基因片段 ,用小鼠白蛋白启动子 /增强子基因片段取代 p HCV- neo4质粒 (含 HCV5′NCR调控荧光素酶基因 )的 CMV启动子 ,构建了一种白蛋白启动子启动转录的 HCV5′NCR调控荧光素酶表达质粒 (p A1 b- HCV) .该质粒能在小鼠肝癌细胞中表达且较小鼠其它癌细胞中表达水平明显增高 ,表明成功地构建了肝特异性表达的 HCV5′NCR调控荧光素酶表达质粒 .该研究为建立肝特异性表达的 HCV5′NCR转基因小鼠模型奠定了基础 ,对评价

肝脏特异性表达人遗传印记基因PEG10转基因载体的构建和鉴定

目的构建小鼠肝脏特异性表达人遗传印记基因PEG10的转基因载体pALB-PEG10-EGFP,为制备转基因小鼠做准备。方法RT-PCR扩增PEG10基因cDNA序列,克隆入T载体进行酶切、测序鉴定,后将其定向克隆至真核表达载体pALB-EGFP中ALB的下游,构建转基因载体pALB-PEG10-EGFP;在Lipofectamine介导下转染L02细胞,经G418抗性筛选,挑选阳性克隆并扩大培养;采用RT-PCR、Western blot、免疫细胞化学等方法分析PEG10在L02细胞中的表达和细胞内定位。结果酶切和测序结果表明pALB-PEG10-EGFP构建成功;稳定转染后的L02表达有PEG10的mRNA及蛋白,且主要定位于胞浆。结论重组体pALB-PEG10-EGFP的构建初步奠定了转基因小鼠制备的基础。

TYLCV-CHI侵染诱导的抗病毒GFP标记的载体构建

以CTAB法提取含有番茄黄化曲叶病毒TYLCV-CHI病原的烟草曲 叶病烟草DNA为模板，扩增该病毒外壳蛋白启动子序列，克隆到pGEM7Z载体上，在启动子下 游连接上能表达毒素蛋白的RNase基因和终止子NOS，最终将构建的基因转移到带有绿色荧光 蛋白GFP的植物表达载体上，构建了通过病毒侵染后激发病毒外壳蛋白启动子从而表达毒素 基因，产生毒素杀死病毒防止感染继续扩散的自我保护机制的基因工程植物表达载体。

稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株的构建

目的构建稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株。方法PCR扩增获得ALB启动子，并与pBGLuc连接获得携带ALB启动子及荧光素酶报告基因的pBGLuc—ALB质粒，脂质体转染质粒到不同细胞，ALB—GLuc活性检测功能。构建逆转录病毒，感染HP14．5肝干细胞株获得携带ALB启动子及荧光素酶报告基因的稳定细胞株，经Dex、HGF体外诱导后第3、6、9、12天ALB—GLuc检测荧光素酶活性，免疫荧光检测ALB的表达。结果PCR、酶切及测序结果显示ALB启动子正确插入至荧光素酶GLuc基因上游，HEK293、HP14．5、LC14d及Hepa1-6细胞中ALB—GLuc活性与免疫荧光结果一致。HP14．5ALB—Gluc稳定细胞株在高浓度的稻瘟菌素中存活，免疫荧光结果显示Dex、HGF诱导后细胞中ALB的表达逐渐增强，并与ALB—Gluc活性升高一致。结论成功构建了稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株，为研究肝干细胞的体外成熟分化提供了重要的细胞手段。

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

Characterization of promoters in Escherichia coli and application for xylitol synthesis

Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-strength inducible promoter with the result that the abnormally high transcription of these genes creates excessive metabolic load on the host, which decreases product formation. The constitutive expression systems are capable of avoiding these defects. In this study, to enrich the application of constitutive promoters in metabolic engineering, four promoters from the glycolytic pathway of E. coli were cloned and characterized using the enhanced green fluorescent protein as reporter. Among these promoters, Pgap Awas determined as the strongest one, the strength of which was about 8.92% of that of the widely used inducible promoter PT7. This promoter was used to control the expression of heterologous xylose reductase in E. coli for xylitol synthesis so as to verify its function in pathway engineering. The maximum xylitol titer(40.6 g·L-1) produced by engineered E. coli under the control of the constitutive promoter Pgap Awas obviously higher than that under the control of the inducible promoter PT7,indicating the feasibility and superiority of promoter Pgap Ain the metabolic engineering of E. coli.

Bioinformatics Analysis of Upland Cotton Gene GhCRE1

Cytokinin plays a very important role in plants growth and development,and CRE1 has been identified as a cytokinin receptor.However,the biological function of CRE1 in cotton remains unclear.In this paper,GhCRE1 gene was cloned and its sequence was analyzed by bioinformatics.The results revealed that GhCRE1 had 11 exons and 10 introns,and its molecular weight,theoretical isoelectric point(pI)and number of amino acids differed from those of the homologous gene in Theobroma cacao and Arabidopsis thaliana,with three different protein domains.15 unidentified proteins were found in potential interaction with CRE1 protein and 2 phosphorylation sites on CRE1 protein sequence were predicted by mutiple bioinformatics websites.Additionally,8 cis-acting regulatory elements were detected on CRE1 promoter sequence,and found related to light signal and hormone.These results were conducive to unfolding the function of GhCRE1 in upland cotton.

Theoretical basis of a beneficial role for vitamin D in viral hepatitis

Abnormal bone metabolism and dysfunction of the calcium-parathyroid hormone-vitamin D axis have been reported in patients with viral hepatitis. Some studies suggested a relationship between vitamin D and viral hepatitis. Genetic studies have provided an opportunity to identify the proteins that link vitamin D to the pathology of viral hepatitis (i.e., the major histocompatibility complex class Ⅱ molecules, the vitamin D receptor, cytochrome P 450 , the renin-angiotensin system, apolipoprotein E, liver X receptor, toll-like receptor, and the proteins regulated by the Sp1 promoter gene). Vitamin D also exerts its effects on viral hepatitis via non-genomic factors, i.e., matrix metalloproteinase, endothelial vascular growth factor, prostaglandins, cyclooxygenase-2, and oxidative stress. In conclusion, vitamin D could have a beneficial role in viral hepatitis. Calcitriol is best used for viral hepatitis because it is the active form of the vitamin D 3 metabolite.

Studies on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled, in part,by the 5’-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development. Using gel mobility shift assays and DNasel footprinting assays, a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ε-globin gene. We speculated that the E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ε-R1 might be an erythroid- and developmental stagespecific repressor. Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.

Relationship between Expression of the Human Alpha-Fetoprotein Gene and DNA Methylation Status of the Promoter Region

OBJECTIVE DNA methylation has been regarded as an important epige-netic signature reflecting the transcription state of DNA in cells. This study was to conducted to assess the relationship between human alpha-feto-protein (AFP) gene expression and the DNA methylation status of the promoter region in three different cells, namely two human hepatocellular carcinoma (HCC) cell lines and normal human fibroblasts. METHODS Transcription of the AFP gene was verified by RT-PCR. After bisulphate treatment of DNA, the methods of MSP and BSP were used to analyze the methylation density and status within single DNA strands of two closely spaced CpG dinucleotides of the promoter region in the different cells. RESULTS RT-PCR analysis indicated that the expression of the AFP gene in HepG2 cells was significantly higher than in SMMC-7721 cells, and that the AFP gene was not expressed in normal human fibroblasts. By MSP and BSP we observed that the promoter region was demethylat-ed in the AFP-high-expressing cell lines, and that the sites of -2,494 bp and -2,431 bp in the AFP genomic sequence can be used as detection sites for early tumorous diagnosis. CONCLUSION These results indicate that the DNA methylation state of the promoter region has a negative correlation with AFP gene expression.

Reactive protein, plasminogen activator inhibitor type-1 （PAI-1） levels, PAI-1 promoter 4G/5G polymorphism and acute myocardial infarction

Objective To investigate the relationship between CRP, plasminogen activator inhibitor type 1 （PAI-1） levels, PAI-1 gene promoter 4G/5G polymorphism and the type of acute myocardial infarction （ST elevation myocardial infarction, STEMI vs the non-ST elevation Myocardial infarction, NSTEMI）. Methods One hundred seventy-six consecutive patients with AMI were included for the study, of whom 60 had STEMI and 56 had NSTEMI, and 60 adults without cardiovascular and cerebrovascular disease were selected as controls. Blood samples were obtained from patients within 6 h of AMI and the plasma PAI-1, CRP, and the gene polymorphism were measured. Results Plasma levels of PAI- 1 and CRP were higher in AMI groups, compared those in the control group, and plasma levels of PAI-1 were significantly higher in patients with STEMI compared to those with NSTEMI （80.12ng/ml VS.73.01ng/ml, P 〈0.01）, while CRP levels were not significantly different between patient with STEMI and NSTEMI （3.87 ± 0.79 mg/ml VS.4.01 ±0.69mg/ml, P〉0.05）. PAI-1 levels presented a significant correlation with CRP levels in the NSTEMI subjects. However, PAI-1 and CRP levels could explain the lack of a significant relationship between them in control and STEMI subjects.The frequencies of 4G/4G genotype in the AMI group were higher than those in the control group and higher in patient with STEMI than in patient with NSTEMI. Plasma levels of PAI-1 in subjects with 4G/4G genotype were significantly increased as compared to those in subjects with 4G/5G and 5G/5G genotype. Conclusions Plasma PAI-1 levels were associated with different myocardial infarction type, and PAI-1 promoter 4G/5G polymorphisms and CRP may be related to plasma PAI-1 levels

A stage-specific protein factor binding to a CACCC motif in both human β-globin gene promoter and 5'-HS2region

The DNasel hypersensitive site 2 (HS2) of human β-globin locus control region (LCR) is required fOr the high level expression of human d-globin genes. In the present study, a stage-specific protein factor (LPF-β) was identified in the nuclear extract prepared from mouse fetal liver at d 18 of gestation, which could bind to the HS2 region of humanβ-globin LCRt We also found that the shift band of LPF-βfactor could be competed by humanβ-globin promoter. However, it couldn’t be competed by human E-globin promoter or by human Aβ-globin promoter. Furthermore, our data demonstrated that the binding-sequence of LPF-d factor is 5’CACACCCTA 3’,which is located at the HS2 region ofβ-LCR (from -10845 to -10853 bp) and humanβ-globin promoter (from -92 to -84 bp). We speculated that these regions containing the CACCC box in both the humallβ-globin promoter and HS2 might function as stage selector elements in the regulation of humanβd-globin switching and the LPF-βfactor might be a stage-specific protein factor involved in the regulation of humanβ-globin gene expression.

A Pin gene families encoding components of auxin efflux carriers in Brassica juncea

Based on the sequence information of Arabidopsis PIN1, two cDNAs encoding PIN homologues from Brassica juncea, Bjpin2 and Bjpin3, were isolated through cDNA library screening. Bjpin2 and Bjpin3 encoded proteins containing 640 and 635 amino acid residues, respectively, which shared 97.5% identities with each other and were highly homologous to Arabidopsis PIN1, PIN2 and other putative PIN proteins. BJPIN2 and BjPIN3 had similar structures as AtPIN proteins. Northern blot analysis indicated that Bjpin2 was expressed in stem, leaf and floral tissues, while Bjpin3 was expressed predominantly in stem and hypocotyls. Two promoter fragments of pin genes, Bjpin-X and Bjpin-Z, were isolated by 'genome walking' technique using primers at 5'-end of pin cDNA. Promoter-gus fusion studies revealed the GUS activities driven by Bjpin-X were at internal side of xylem and petal; while those driven by Bjpin-Z were detected at leaf vein, epidermal cell and cortex of stem, vascular tissues and anther. Results of the pin genes with different expression patterns in B. juncea suggested the presence of a gene family.

Identification of the development stage-specific factors in mouse fetal liver binding to the human β-globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By using DNase I footprinting and gel mobility shift assays, the binding of protein factors in these extracts to the human βglobin promoter was analyzed. The differences in the binding patterns of protein factors during development were observed. An erythroid-specific and stage-specific nuclear protein in the nuclear extract from d 18 mouse fetal liver was identified, which can bind to the sequence (from -66bp to -90bp) of human β-globin promoter. We therefore speculate that the function of this cis-acting element may be similar to stage selector element (SSE) in chieken βA- promoter.

Functional properties of a chitinase promoter from cabbage (Brassica oleracea var. capitata)

The 5’-region of the chitinase gene cabch29, derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5’-deletion fragments were linked to reporter geneβ-glucuronidase (GUS) as translational fusions, and the expression of these chimeric genes was analyzed in vegetative organs and tissues. Sequences up to -651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues. The addition of further upstream sequences (-651 to -1284) enhanced expression level, and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding. Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-GUS fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment. The location of GUS activity appears to be cell-specific, being highest in vascular cells and epidermal cells of stem, leaf and roots. Meanwhile, the temporal and spatial expression of cabch29-GUS fusion gene has been investigated. Among the different vegetative organs, a high level of GUS activity was observed in stem and a moderate one in roots;whereas, wounding stress led to a high level of GUS in stem and moderate one in leaf.

Metabolomics Study in Methylotrophic Yeast： A Minireview

Methylotrophic yeast has been used as a cost-effective and valuable host for expression of recombinant protein due to its unique methanol utilisation pathway. It has an AOX （alcohol oxidase） protein which has been characterised to be a strong and tightly methanol-inducible dependent promoter. Metabolomics is the systematic study and inclusive analysis of small molecules called metabolites in a biological system. Metabolomics plays an important part in connecting the phenotype and genotype gap because it magnifies the modifications in the proteome and provides a better phenotype representation of an organism. This quantitative study has provided a new perception on the metabolic burden derived from the overexpression of recombinant protein in methylotrophic yeast. In this review, we discuss the fundamental aspect of metabolomics in methylotrophic yeast followed by their latest developments.

甜叶菊浸膏对抑郁小鼠脾脏组织中DBP和SP蛋白表达的影响

目的探讨甜叶菊浸膏的抗抑郁活性及其对脾脏和大脑皮层组织中白蛋白启动子D位点结合蛋白(DBP)与P物质(SP)蛋白表达的影响。方法将60只BALB/c♂小鼠随机均分为对照组、模型组、阳性对照组、甜叶菊浸膏低剂量组(SEL,1.5 g·kg^(-1))和高剂量组(SEH,3 g·kg^(-1))。用皮质酮悬液按20 mg·kg^(-1)剂量连续于腹腔注射21 d,复制小鼠抑郁模型,每天给药1次。待造模成功后,通过ig途径给予药物治疗。正常对照组和模型组继续给予生理盐水,阳性对照组给予氟西汀(30 mg·kg^(-1)),甜叶菊浸膏各组给予相应剂量的药物,连续给药7 d,每天给药1次。用旷场法测定各组小鼠的平行移动格数和直立次数等行为学指标;用Western Blot技术测定小鼠脾脏与大脑皮质中DBP和SP蛋白的表达。结果模型组小鼠的平行移动格数和直立次数明显减少,与对照组的比较有显著性差异(P<0.01),提示模型建立成功。给予甜叶菊浸膏后,可剂量依赖性地增加抑郁小鼠的平行移动格数(SEL:54.17±13.33,SEH:73.17±13.90)和直立次数(SEL:35.42±5.12,SEH:54.83±11.60),与模型组的比较有显著性差异(移动格数:20.08±3.53,直立次数:9.33±1.83);甜叶菊浸膏显著地增加了脾脏和脑组织中SP蛋白的表达水平,与模型组的比较有显著性差异(P<0.05,P<0.01);甜叶菊浸膏减少了脾脏组织中DBP蛋白的表达,却增加了脑组织中DBP蛋白的水平,与模型组的比较有显著性差异(P<0.01)。结论甜叶菊浸膏可有效改善小鼠的抑郁症状,其抗抑郁机制可能与调控大脑皮层和脾脏组织中DBP及SP蛋白的表达水平有关。

Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells

Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results: Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion: Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.