人era真核表达载体的构建

Era是一个独特的含有RNA结合KH结构域的G蛋白亚家族,哺乳类era新近被克隆,目前还没有关于其功能的研究报道。构建了带流感病毒血凝素9肽表位标签（HA tag）的野生型人era基因的真核表达载体,瞬时转染体外培养细胞CHO,Western blot鉴定表明目的的基因得到表达。为深入探讨人era基因的功能奠定了坚实的基础。     

中国人肥胖基因真核表达载体的构建

为研究肥胖（ｏｂｅｓｉｔｙ）的病因及肥胖基因（ｏｂ）的表达与调控,根据文献报道的ｈｏｂ序列设计引物,经ＲＴ－ＰＣＲ扩增中国人的ｏｂ基因（包括信号肽在内的ｃＤＮＡ全长５４０ｂｐ）,ＰＣＲ产物采用Ｔ－Ａ克隆法首先连接到克隆载体ｐＵＣ１１９,然后定向转移到经改造的真核表达载体ｐＳＶ－β－ｌａｃＺ,酶谱分析表达克隆基因为的人肥胖基因。     

hCGβ真核表达载体的构建和瞬时表达筛选

为探讨人绒毛膜促性腺激素β亚基（hCGβ）基因避孕疫苗的可能性,利用DNA重组技术将hCGβ的基因片段连接到真核表达载体pCMV4上,酶切分析鉴定正确构建了质粒DNApCMV4-hCGβ,将质粒DNApCMV4-hCGβ通过脂质体转染的方法导入体外培养的Hela细胞,双抗夹心ELISA法检测质粒DNApCMV4-hCGβ在Hela细胞瞬时表达系统中特异性的表达了hCGβ。结果表明hCGβ表达含量24h为6.78ng/ml,48h为15.24ng/ml,说明在体外瞬时表达了特异性hCGβ的pCMV4-hCGβ真核表达载体能够作为DNA疫苗使用,为pCMV4-hCGβ质粒DNA免疫小鼠进行DNA避孕疫苗的研究奠定了基础。     

中国株HIV-1核心蛋白真核表达载体的构建与表达

运用限制性内切酶XbaⅠ、SalⅠ对pKSGAG进行双酶切,获得HIV-lgag基因,并与真核表达载体pCI—neo连接,构建含有中国流行株HIV-1核心蛋白真核表达载体pCI—neoGAG。经XbaⅠ／SalⅠ双酶切及测序鉴定证实,成功地构建了HIV-1核心蛋白真核表达载体pCI-neoGAG。通过脂质体将pCI—neoGAG转染入p815细胞,G418筛选4周后,使用间接免疫荧光方法检测表达产物。结果表明所构建的HIV-1核心蛋白真核表达载体能在p815细胞中高效表达,为下一步进行HIV-1 DNA疫苗研究奠定了基础。     

插入CpG序列的汉坦病毒S基因真核表达载体的构建及表达

To improve the effect of the gene immunization against Hantaan virus, we constructed the eukaryotic expression vector pTARGET-hans(ISS) containing Hantaan Virus S gene coding region and CpG motif by cloning S gene segment with CpG motif into eukaryotic expression vector pTARGET^TM.After conformed by enzyme analysis, the recombinant expression vector pTARGET-hans(ISS) was transferred into Vero-E6 cells by electroporation and the transient expression of Hantaan virus nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transferred Vero-E6, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector pTARGET-hans(ISS) was successfully constructed and expressed in vitro,which will lay a foundation for further animal vaccination.     

中国株HIV-1外膜蛋白真核表达载体的构建与表达

To construct the eukaryotic expression vector of HIV-1 gp120 gene and observe its expression in vitro, the recombinant expression vector pVAX1GP120 was constructed by inserting the gp120 gene into the eukaryotic expression vector pVAX1. The pVAX1GP120 was transfected into Vero cells by lipofectamine and the expressed product was detected by indirect immunofluore- scence.Restriction enzymes digestion analysis and sequencing results revealed that the recombinant expression vector pVAX1GP120 has been constructed successfully. The indirect immunofluorescence result showed green fluorescence on the membrane of transfected cells. The constructed eukaryotic expression vector of HIV-1 gp120 can be expressed in vitro, which lay the foundation for the further study of HIV-1 DNA vaccine.     

猪圆环病毒2型ORF2编码基因真核表达载体的构建及表达

根据GenBank中猪圆环病毒2型(PCV-2)ORF2基因序列,设计了1对引物,应用PCR从含有PCV-2的PK-15细胞中扩增出ORF2基因,将其克隆入pSecTag2载体中,构建了pSecORF2载体。又设计一条含信号肽序列的上游引物,以pSecORF2为模板,扩增出含信号肽序列的ORF2基因,将其克隆到pIREShyg载体上,构建了pIRESiORF2真核表达载体。然后通过磷酸钙共沉淀法转染CHO细胞,进行表达。间接免疫荧光实验(IFA)成功检测到pIRESiORF2在CHO细胞中的表达。这为进一步研究ORF2编码蛋白的生物学活性及建立PCV诊断试剂盒打下基础。     

Angiostatin基因真核表达载体的构建及对B16黑色素瘤体内生长抑制作用

Angiostatin是一种新发现的对肿瘤生长有特异抑制作用的抗血管生成因子,实验已证实其对多种肿瘤有明显的抑制作用。本文报告构建了含Angiostation基因的真核表达载体pAG3,通过建立荷瘤小鼠模型来研究Angiostation对人黑色素瘤B16的原位生长,植入及与化疗药物DTIC的联合作用等来探讨Angiostation裸DNA肌肉注射的体内抗瘤效应。实验结果表明Angiostation可明显抑制C57荷瘤小鼠的肿瘤生长;人黑色素瘤B16细胞植入前5天肌肉注射pAG3能显著阻止下C57小鼠新肿瘤的形成;但在pAG3与DTIC联合化疗实验中,两者未表现出明显的增强效应。本实验为拓展非病毒介导的Angiostation抗血管生成基因治疗途径奠定了基础。     

IBDV多聚蛋白基因真核表达质粒的构建与表达

将IBDV上海超强毒株的多聚蛋白基因(vp2-4-3)克隆入真核表达载体pALTER-MAX,构建成功pALTER-MAX-VP2-4-3真核表达质粒,经纯化后,pALTER-MAX-VP2-4-3在Lipofectamie^TM2000介导下转染Vero细胞、11日龄鸡胚的绒毛尿囊膜(CAM)和肌肉注射2日龄的雏鸡,1周后,分别提取细胞或组织中的总DNA或总RNA,用DIG标记探针均可检测到阳性杂交信号;转染的Vero细胞飞片和肌肉冰冻切片,进行免疫荧光检测均呈现阳性结果;转染的鸡胚CAM匀浆上清,用兔抗IBDV超强毒的高免血清,经Dot—ELISA检测呈现阳性。表明转染后基因获得表达,表达的蛋白具有免疫反应性。     

HCV全长基因真核表达载体的构建及其细胞内表达

本实验成功构建了HCV全长基因的真核表达载体pCI-HCVFL,转染HepG2细胞后经免疫荧光和免疫组化法分别检测到结构基因区(C)和非结构基因区(NS3)病毒蛋白的表达,该载体可用于建立HCV转基因细胞模型以及进一步开展有关HCV复制与表达的深入研究。     

Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund’s Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

乙型肝炎病毒e抗原基因与绿色荧光蛋白基因的融合及其表达

质粒pS65T含有T7启动子驱动的绿色荧光蛋白突变型gfpS65T基因,经修饰后,在其中插入乙型肝炎病毒e抗原（HBeAg）基因,使两基因同框成为融合基因。在大肠杆菌BL21中由于T7启动子的控制,高效表达了具有双功能（抗原性和发光性）的融合蛋白（GFP－HBeAg）。融合蛋白MW为52kDa,N端有6个组氨酸残基,因而用Ni金属螯合层析柱对融合蛋白进行了分离纯化,利用诊断HBV的ELISA试剂盒检测了融合蛋白的抗原性,在荧光显微镜下观察到了融合蛋白的绿色荧光。并探讨了用其组装成新型免疫诊断试剂的可能性。     

新型瞬时报告载体pRSET-EGFP的构建及表达

目的：利用分子生物学技术和方法将pRSET-B质粒改建为带有绿色荧光蛋白(GFP)突变体基因(GFP-S65T)的新型瞬时表达载体pRSET-EGFP,并在E．coli．BL21中得到GFP基因的高效表达。方法：PCR法从pEGFP质粒克隆GFP-S65T cDNA并在5’末端引入KpnⅠ的位点。将扩增出来的GFP-S65T基因和pRSEY-B质粒用HindⅢ和KpnⅠ双酶切后连接构成重组质粒。用化学法把重组质粒转化到E．coli．BL21中,培养发酵液。OD540=0．4时加入IPTG诱导GFP-S65T基因转录和表达,合成绿色荧光蛋白。还对诱导条件进行了优化,发酵液OD540=0．4加入IPTG可以得到最优表达。结果：通过Ni^2 柱亲和层析,纯化得到绿色荧光蛋白,SDS-PAGE电泳检测,分子量为27kDa,与文献报道值一致。这说明Ni^2 柱能够有效的纯化表达产物。结论：成功构建了新型瞬时表达载体pRSET-EGFP,并且在E．coli．BL21中得到高效表达。     

Characterization of the cDNAs Encoding Three GnRH Forms in the Pejerrey Fish <Emphasis Type="Italic">Odontesthes bonariensis</Emphasis> (Atheriniformes) and the Evolution of GnRH Precursors

Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A “forebrain lineage” separates evolutionarily from the “midbrain lineage” as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates. [Reviewing Editor: Dr. Axel Meyer]     

携带GFP发光基因人猴嵌合免疫缺陷病毒SHIV的构建及活性检测

目的获得正常感染宿主细胞并稳定表达绿色荧光的SHIV毒株,为后期建立发光SHIV/恒河猴感染模型奠定基础。方法通过分子克隆手段,将绿色荧光蛋白基因克隆到携带HIV-1包膜蛋白的SHIV病毒全基因组中,并在细胞水平检测各毒株的感染活性及荧光蛋白表达能力。结果得到一株可表达绿色荧光蛋白的病毒株SHIV-KB9nefGFP,并具有感染TZM-bl细胞系及猴PBMC的能力。结论该毒株在宿主细胞恒河猴PBMC中具有一定复制能力,希望通过后续的猴体内传代实验获得毒力更强的发光病毒。     

GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

GnRH类似物对子宫肌瘤大鼠模型血液黏度、子宫系数和炎性细胞浸润的影响

摘要 目的：探讨促性腺激素释放激素（Gonadotropin releasing hormone,GnRH）类似物对子宫肌瘤大鼠模型血液黏度、子宫系数和炎性细胞浸润的影响。方法：子宫肌瘤大鼠模型（n=36）随机平分为三组-模型组、米非司酮组与GnRH类似物组,分别给予腹腔注射0.15 ml的生理盐水、2 mg/kg米非司酮与2 mg/kg GnRH类似物,每周1次,持续8周。结果：米非司酮组与GnRH类似物组治疗第4周与第8周的全血比黏度、卵泡刺激素（FSH）、黄体生成素（LH）值都低于模型组（P<0.05）,GnRH类似物组低于米非司酮组（P<0.05）。米非司酮组与GnRH类似物组治疗第8周的子宫系数、子宫白介素（IL）-10与肿瘤坏死因子（TNF）-α表达水平都低于模型组（P<0.05）,GnRH类似物组低于米非司酮组（P<0.05）。米非司酮组与GnRH类似物组治疗第8周的子宫内膜厚度、腺间质面积比、腺体面积与腺腔面积都高于模型组（P<0.05）,GnRH类似物组高于米非司酮组（P<0.05）。米非司酮组与GnRH类似物组治疗第8周的子宫组织Wnt5b与β-catenin蛋白相对表达水平低于模型组（P<0.05）,GnRH类似物组低于米非司酮组（P<0.05）。结论：GnRH类似物在子宫肌瘤大鼠模型的应用能降低子血液黏度,还可抑制血清性激素的分泌,增加子宫系数,抑制Wnt5b与β-catenin蛋白的表达,从而可改善子宫肌瘤大鼠的炎性细胞浸润状态与子宫内膜形态。     

慢病毒载体介导GHRH基因在小鼠肌肉组织表达及对动物生长的影响

人与动物体内生长激素受生长激素释放激素(Growth Hormone Releasing Hormone,GHRH)与生长激素抑制激素(Somatostatin,SST)两种因子共同调节,在体内表达外源GHRH,可以提高体内GH基础水平,进而达到促进体内GH释放,加速动物生长的效果.对慢病毒载体系统加以改造,使之成为C...     

Expression of estrogen receptor alpha and beta in myometrium of premenopausal and postmenopausal women

Although a clear role for estrogen receptor (ER) alpha has been established, the contribution of ERbeta in estrogen-dependent development, growth and functions of the myometrium is not understood. As a first step towards understanding the role of ERbeta, we have examined the expression of ERalpha and ERbeta in the human myometrium. With competitive RT-PCR assays, the level of ERbeta mRNA was 10-200 times lower than that of ERalpha mRNA in both premenopausal and postmenopausal myometrium. In premenopausal myometrium, the expression pattern of ERbeta mRNA during the menstrual cycle was similar to that of ERalpha mRNA, with highest levels in peri-ovulatory phase. In postmenopausal myometrium, ERbeta mRNA was significantly higher than it was in premenopausal myometrium, while the level of ERalpha mRNA was lower. The net result was a change in the ratio of ERbeta to ERalpha mRNA expression. The ratio changed from 0.6-1.5 in premenopausal to 2.5-7.6 in postmenopausal myometrium. In premenopausal women, the gonadotropin releasing hormone analogue, leuprorelin acetate, elicited a decrease in ERalpha and an increase in ERbeta mRNA expression to cause a postmenopausal receptor phenotype. Estradiol, on the other hand, reversed ERalpha and ERbeta mRNA expression and their ratio in postmenopausal myometrium to those of premenopausal myometrium. Immunohistochemical staining and Western blot analysis of ERalpha and ERbeta with semiquantitative analysis showed good agreement between mRNA and protein levels. The data indicate that coordinated expression of ERalpha and ERbeta might be necessary for normal estrogen action in myometrium. Furthermore, estrogen appears a dominant regulator of both receptors in the myometrium.