Kozak序列+4G提高绿色荧光蛋白在HEK293细胞中的表达

构建含不同Kozak序列的绿色荧光蛋白(GFP)基因真核表达载体, 并检测它们在HEK293细胞中的表达差异。 通过设计突变的PCR引物改变目的基因GFP的Kozak序列, +4 位碱基分别为A和G, 且不改变氨基酸编码, 将PCR扩增的GFP片段与载体pcDNA3.1进行酶切、连接、转化、鉴定。成功构建的pHGFP-A, pHGFP-G质粒采用脂质体法转染HEK293细胞, 荧光显微镜下观察绿色荧光表达, 流式细胞术检测目的蛋白GFP的荧光表达阳性率, Western blot检测目的蛋白GFP的表达。构建的两质粒均能有效转染 HEK293细胞, 其中流式细胞术分析显示: pHGFP-A组GFP阳性率约为15%, pHGFP-G组GFP阳性率约为45%; Western blot 显示pHGFP-G的GFP表达量约为pHGFP-A的GFP表达量3.87倍。结果表明, Kozak序列+4G(?3位为嘌呤碱基时)在蛋白表达中发挥重要作用, 可以使绿色荧光蛋白GFP在HEK293细胞中的表达量提高约4倍。     

Kozak序列+4G提高绿色荧光蛋白在HEK293细胞中的表达

构建含不同Kozak序列的绿色荧光蛋白(GFP)基因真核表达载体, 并检测它们在HEK293细胞中的表达差异。 通过设计突变的PCR引物改变目的基因GFP的Kozak序列, +4 位碱基分别为A和G, 且不改变氨基酸编码, 将PCR扩增的GFP片段与载体pcDNA3.1进行酶切、连接、转化、鉴定。成功构建的pHGFP-A, pHGFP-G质粒采用脂质体法转染HEK293细胞, 荧光显微镜下观察绿色荧光表达, 流式细胞术检测目的蛋白GFP的荧光表达阳性率, Western blot检测目的蛋白GFP的表达。构建的两质粒均能有效转染 HEK293细胞, 其中流式细胞术分析显示: pHGFP-A组GFP阳性率约为15%, pHGFP-G组GFP阳性率约为45%; Western blot 显示pHGFP-G的GFP表达量约为pHGFP-A的GFP表达量3.87倍。结果表明, Kozak序列+4G(?3位为嘌呤碱基时)在蛋白表达中发挥重要作用, 可以使绿色荧光蛋白GFP在HEK293细胞中的表达量提高约4倍。     

用绿色荧光蛋白（GFP）作为报告分子筛选有效的siRNA

 建立一种利用绿色荧光蛋白（GFP）作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子（MIF）基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.     

猪β-内啡肽基因克隆和重组质粒壳聚糖修饰分子包装体外表达研究

目的克隆猪β-内啡肽基因,研究其体外原核、真核表达,探索高效表达目的基因的新型纳米材料,为进一步研究β-内啡肽对动物免疫应答和应激调节等方面的作用奠定基础。方法利用基因延伸重叠PCR克隆β-内啡肽的基因cDNA,双酶切后插入VR1020、pGEX-4T-1,构建其真核、原核表达载体;以不同浓度的IPTG诱导表达重组融合蛋白,并作SDS-PAGE和RT-PCR分析目的基因原核表达情况;利用壳聚糖及其修饰分子制备纳米颗粒包装重组真核表达质粒,体外转染HEK293细胞,提取总RNA进行RT-PCR分析β-内啡肽基因在真核细胞的表达。结果成功克隆了猪β-内啡肽基因,并构建获得其原核和真核表达载体;不同浓度的IPTG诱导后,在29.7kDa的位置出现了新的蛋白条带,而原来的26.2kDa处的GST标签蛋白条带消失。说明目的基因已经和GST融合产生了新的融合蛋白;壳聚糖及其聚乙二醇和聚乙烯亚胺接枝修饰分子(mPEG-PEI-CS)纳米颗粒包裹VREP转染HEK293细胞,48h后收集细胞,RT-PCR和ELISA检测显示VREP能够在HEK293细胞中高效地转录表达β-内啡肽。结论本实验成功克隆了猪的β-内啡肽基因,并成功进行了原核及真核细胞表达分析,壳聚糖修饰分子纳米颗粒包装可高效表达目的基因,为进一步的动物实验奠定了基础。     

多核酶表达系统在HEK293细胞中对多药耐药相关蛋白（MRP1）的表达抑制作用

为了研究多核酶表达系统在HEK293细胞中对多药耐药相关蛋白表达抑制的作用．我们构建了含有20个可以自身切割的顺式作用核酶和10个靶向MRP1基因特定位点的反式作用核酶的多核酶表达系统。利用RT—PCR、Westem blot和MTT分析了多核酶系统分别与MRP1靶基因质粒和MRP1 全长基因质粒共转染的HEK293细胞。结果显示．多核酶表达系统能够明显降低荧光融合蛋白在HEK293细胞中的表达。RT—PCR分析表明．懈用靶mRNA降低程度与多核酶表达系统所含的反式作用核酶数目有关。Westernblot分析显示了与RT—PCR相似的结果。Mrrr分析表明,多核酶表达系统能够逆转由MRP1基因转染HEK293细胞产生的多药耐药性。结果提示．含有多个核酶的表达系统对MRP1基因的抑制效应优于单核酶的表达系统。因此．该策略可能用于基因治疗肿瘤或其他疾病．     

人源跨膜蛋白43(TMEM43)的检测、细胞内定位及体外真核表达

为探究人源跨膜蛋白43(TMEM43)基因含量、定位分布及体外真核表达情况,利用RT-PCR及Western blot方法分别检测内源MRC-5和HEK293细胞内TMEM43基因及其蛋白表达情况,采用间接免疫荧光实验进一步分析其在MRC-5细胞内的分布;构建重组表达载体pcDNA3.1-EGFP-TMEM43,转染至HEK293细胞内检测外源TMEM43在HEK293细胞内的基因和蛋白表达情况。结果显示,基因水平TMEM43在MRC-5细胞内的表达明显高于HEK293细胞,蛋白水平仅能检测到MRC-5细胞内TMEM43的表达。重组表达质粒pcDNA3.1-EGFP-TMEM43转染HEK293细胞后,基因水平TMEM43表达显著升高, Western blot可检测到转染后细胞内TMEM43蛋白成功表达。结果表明,不同细胞表达TMEM43含量存在差异,肺来源细胞内TMEM43的表达明显高于肾来源的细胞;构建的重组载体pcDNA3.1-EGFP-TMEM43可成功转染HEK293细胞并表达。该研究为TMEM43结构及功能、与疾病相关机理的进一步探究提供了实验依据。     

肿瘤细胞对血清补体杀伤耐受的机制研究

目的:探讨肿瘤细胞耐受血清补体杀伤的分子机制。方法:将表达不同血型抗原的肿瘤细胞与添加同血型血清的培养基孵育,获得耐受血清杀伤的肿瘤细胞。无血清悬浮培养肿瘤干细胞,对耐受血清杀伤的肿瘤细胞和肿瘤干细胞分别提取mRNA,进行反转录PCR,检测肿瘤干细胞干性标志物和补体调节蛋白的表达情况;干扰补体调节蛋白,检测肿瘤细胞对血清补体的杀伤情况。结果:对分别表达血型抗原A、B和H的HT-29、KATOⅢ、MCF7肿瘤细胞均获得耐受血清杀伤的耐受细胞,耐受细胞高表达三种膜性补体调节蛋白CD46、CD55和CD59,以及部分肿瘤干细胞干性标志物;spheroid悬浮球肿瘤干细胞高表达上述三种膜性补体调节蛋白,特别是CD46;在肿瘤细胞中腺病毒干扰CD46的表达,可显著增强血清补体对肿瘤细胞的杀伤率。结论:补体调节蛋白CD46分子可通过增强肿瘤细胞的干性而介导肿瘤细胞对血清补体的杀伤耐受。     

人Rab12基因真核表达载体的构建及在HEK293细胞中的表达

Rab蛋白参与细胞的囊泡运输过程。自噬体-溶酶体的融合需要活化的Rab12蛋白参与。本研究从HEK293细胞获取总RNA,采用RT-PCR方法扩增出Rab12基因的ORF全序列,将其克隆到含有增强型绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-N1上,成功构建重组质粒pEGFP-Rab12,然后采用lipofectamine 2000将重组质粒转染至HEK293细胞中并获得高表达。     

USP22基因克隆及其融合蛋白真核表达载体的构建

目的克隆人类泛素水解酶22（ubiquitin—specific processing enzyme22,USP22）基因,构建与绿色荧光蛋白融合表达的真核载体。方法利用RT—PCR技术以HeLa细胞总RNA为模板分别扩增USP22基因cDNA序列两片段,依次插入真核表达载体pEGFP—N1;重组质粒转染HEK293T细胞,观察融合蛋白表达及绿色荧光的分布。结果测序结果显示USP22序列与GenBank收录数据一致,酶切显示重组质粒pEG—FP—USP22构建无误,质粒转染后有80％左右HEK293T细胞表达绿色荧光,且在胞质与胞核中广泛分布。结论成功克隆USP22基因并构建与GFP融合表达的真核载体,为进一步深入研究USP22基因的生物学功能奠定了基础。     

外源表达的人copine Ⅴ诱导细胞死亡的研究

目的:copines蛋白家族是新近发现的一类分布广泛、进化保守的Ca2 依赖性磷脂结合蛋白,copineⅤ是其成员之一。为研究copineⅤ基因的功能,构建了人copineⅤ基因的真核表达质粒并转染HEK293细胞,以进一步研究其功能。方法:构建人copineⅤ编码区序列的真核表达质粒pCDNA4/copineⅤ,脂质体法转染HEK293细胞。通过RT-PCR、克隆形成实验和Hochesst33342染色等方法观察copineⅤ基因对HEK293细胞生长的影响。结果:外源表达的人copineⅤ可以诱导HEK293细胞死亡,无法筛选到稳定克隆。结论:人copineⅤ可以诱导HEK293细胞死亡,为进一步研究copineⅤ基因的生物功能提供了线索。     

Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.     

Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

Evidence of complement-mediated killing of Discocotyle sagittata (Platyhelminthes, Monogenea) oncomiracidia

Discocotyle sagittata oncomiracidia were rapidly killed when incubated in na?ve plasma and immune sera from both rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), the killing proceeding at a faster rate with blood material from the latter fish species. The lethal activity of na?ve plasma and immune sera was comparable. This was abolished after incubation at 45 degrees C for 30 min and by the addition of EDTA but not EGTA supplemented with Mg(2+), indicating that complement acting via the alternative pathway is responsible for the parasiticidal effect observed. Scanning electron micrographs showed varying degrees of surface disruption in larvae exposed to fish plasma, suggesting that complement acts by breaching the oncomiracidial tegument. Control (untreated) oncomiracidia showed no damage. Ultrastructural damage was more extensive in oncomiracidia exposed to brown trout plasma than to rainbow trout plasma for equal periods, suggesting that the complement cascade may be involved in mediating host susceptibility.     

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

The vitronectin gene in rainbow trout: cloning, expression and phylogenetic analysis

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon-intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.     

Negative regulation of MEKK1-induced signaling by glutathione S-transferase Mu

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) is an important component in the stress-activated protein kinase pathway. Glutathione S-transferase Mu 1-1 (GST M1-1) has now been shown to inhibit the stimulation of MEKK1 activity induced by cellular stresses such as UV and hydrogen peroxide. GST M1-1 inhibited MEKK1 activation in a manner independent of its glutathione-conjugating catalytic activity. In vitro binding and kinase assays revealed that GST M1-1 directly bound MEKK1 and inhibited its kinase activity. Co-immunoprecipitation analysis showed a physical association between endogenous GST M1-1 and endogenous MEKK1 in L929 cells. Overexpressed GST M1-1 interfered with the binding of MEKK1 to SEK1 in transfected HEK293 cells. Furthermore, GST M1-1 suppressed MEKK1-mediated apoptosis. Taken together, our results suggest that GST M1-1 functions as a negative regulator of MEKK1.