缩短5′-UTR序列可以提高hGH基因在昆虫细胞中的表达

人生长激素是一种由大脑基部垂体前叶分泌合成的多肽类激素 ,其分子量为 2 1 5ｋＤ ,由 191个氨基酸组成。生长激素的功能非常广泛 ,它能刺激骨和软骨的生长 ,器官的发育 ,它还具有维持人体正常代谢 ,如促进ＤＮＡ、ＲＮＡ和蛋白质合成 ,分解脂肪 ,提高血糖水平 ,增加基础代谢 ,改善免疫功能和保护健康组织等功能[1 ] 。Ｂａｃ ｔｏ Ｂａｃ系统是最新发展的昆虫细胞 杆状病毒表达系统之一[2 ] ,是利用转移载体ｐＦａｓｔＢａｃ1转化含有杆状病毒穿梭载体Ｂａｃｍｉｄ的大肠杆菌ＤＨ10Ｂａｃ ,外源基因通过位点特异性转座作用整合到Ｂａｃｍ…     

缩短5＇-UTR序列可以提高hGH基因在昆虫细胞中的表达

人生长激素是一种由大脑基部垂体前叶分泌合成的多肽类激素,其分子量为21.5kD,由191个氨基酸组成.生长激素的功能非常广泛,它能刺激骨和软骨的生长,器官的发育,它还具有维持人体正常代谢,如促进DNA、RNA和蛋白质合成,分解脂肪,提高血糖水平,增加基础代谢,改善免疫功能和保护健康组织等功能[1].     

伪狂犬病毒gE基因在昆虫细胞中的高效表达

In order to develop a simple and safe test for the detection of vaccinated as well as wild type Pseudorabies virus (PRV) infected pigs, the modified gE gene of PRV Ea strain, obtained by cutting the 5' UTR using PCR and DNA recombinant technique, was inserted into baculovirus expression vector pFastBac 1, resulting the trans-position plamid pFE1.75. After homologous recombination, recombinant baculovirus rvBacE1.75 was gained and high level expression of glycoprotein E (gE) was observed after the infection of rvBacE1.75 to Tn-5B1-4 cells. The expression product was 80-88 kD and was specific to antisera against PRV Ea strain by Western-blotting. Purified recombinant proteins were used as an antigen in Latex Agglutination Test(gE-LAT) and the test was specific, sensitive, safe and simple.     

人粒细胞集落刺激因子（hG－CSF）cDNA3′－UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ３′端非翻译区（３′－ＵＴＲ）中存在的ＤｒａⅠ酶切位点,通过部分酶切与完全酶切,删除３′－ＵＴＲ不同长度,构建了四种ｈＧ－ＣＳＦｃＤＮＡ瞬时重组表达质粒。转染ＣＯＳ－７细胞后,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３′－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３′－ＵＴＲ对ｈＧ－ＣＳＦｃＤＮＡ表达的影响与转录水平的差别有一定关系。     

人GDNF基因在昆虫细胞中的高效表达

应用昆虫杆状病毒表达系统在昆虫细胞Ｔｎ－５Ｂ１－４中高效表达了人胶质细胞源性神经营养因子（ＧＤＮＦ）,ＰＡＧＥ分析表达量占细胞可溶性蛋白质的３０％左右,表达产物经亲和层析纯化后纯度达８０％以上,活性研究表明,昆虫细胞表达的ＧＤＮＦ蛋白能显著促进多巴胺能神经元的存活,此研究为进一步研究ＧＤＮＦ结构与功能打下了良好的基础。     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus-infected insect cells. This 21 bp sequence element is derived from a 5' untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20- and seven-fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A-rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus-based expression system.     

重组鸡γ_干扰素在昆虫细胞中的高效表达

将鸡γ-干扰素(ChIFN-γ)基因克隆到载体pFASTBAC1中,构建转移载体pFASTBAC1-ChIFN-γ,然后转化DH10Bac感受态大肠杆菌,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid-ChIFN-γ;通过脂质体将表达质粒转染Sf9昆虫细胞,用间接免疫荧光试验鉴定重组鸡γ-干扰素(rChIFN-γ)的表达;通过水泡性口炎病毒感染鸡胚成纤维细胞(CEF)的细胞病变抑制试验,检测rChIFN-γ的活性。研究结果表明,感染重组杆状病毒的Sf9细胞能高效表达rChIFN-γ,且当每个细胞的感染量为1个病毒时,细胞在感染96h后,rChIFN-γ基因表达产物的活性最高,达到106~107.2U/mL。以rChIFN-γ进行对新城疫病毒(NDV)F48E8株、禽流感H5N1病毒(AIV-H5)和马立克氏病病毒(MDV)GA株的抗病毒活性试验,发现rChIFN-γ对AIV-H5和MDV GA株病毒有明显的抑制其致细胞病变作用,但对NDVF48E8株病毒在体外不能抑制其致细胞病变作用,仅能在病毒滴度上表现抑制效果。     

Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells

A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.     

Expression of the mouse serotonin receptor (5HT1c) cDNA in cultured insect cells

Baculovirus-mediated cloning and expression of the mouse serotonin receptor (5HT1c) cDNA in insect cells was proposed to obtain an alternative to an oocyte-based system, which is commonly employed in electrophysiological studies of ionic channels. A recombinant bacmid was constructed, and the 5HT1c cDNA transferred into the AcNPV genome to yield a recombinant baculovirus. Infected inset Sf9 cells produced recombinant 5HT1c.     

Mu-opioid receptor expression in High Five insect cells is regulated by 5' untranslated region (5'UTR)

There is increasing evidence that the 5'UTR of mRNAs affects regulation of gene expression in eukaryotic cells. We examined the overexpression of the mu-opioid receptor in High Five insect cells, employing rat mu-receptor cDNA linked to variable lenghts of their native 5'UTR. The sequences employed consist of either 209 nucleotides (termed ,,long") upstream the translation initiation site of the mu-receptor mRNA, or a truncated 5'UTR comprising only 11 nucleotides (,,short"). These constructs served to generate recombinant baculovirus for the expression of mu-receptor protein in High Five insect cells. 48 hours after baculovirus infection cells were harvested for mu-receptor characterization or RNA analysis. Scatchard analysis of radioligand binding consistently revealed three to four fold higher concentrations of the mu-opioid receptors expressed with the ,,long" over the ,,short" UTR containing baculovirus. The distinct expression rates of mu-receptors paralleled the amounts of mRNAs determined by RNase protection assay. Regardless of the distinct 5'UTR regions, the expressed opioid receptors displayed identical high affinity binding characteristics for the opioid antagonist diprenorphine and similar EC50 values to inhibit forskolin (10(-5) M) stimulated cAMP synthesis. Our results demonstrate that the native 5'UTR of the mu-opioid receptor has an enhancing effect on expression in the baculovirus/insect cell system.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2′-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.     

Expression of human erythropoietin cDNA in human lymphoblastoid Namalwa cells: the inconsistency of a stable expression level with transient expression efficiency

Recombinant plasmids for the expression of human erythropoietin (EPO) cDNA in Namalwa cells were constructed. From the results of the EPO expression efficiency in transiently transfected cells, it was found that the simian virus 40 (SV40) early promoter directs EPO synthesis more efficiently in Namalwa cells than does the long terminal repeat promoter of Rous sarcoma virus and that the 3'-noncoding sequence including splice junction and polyadenylation site derived from the rabbit beta-globin gene are more effective than those of the SV40 early gene. However, in stable transformants, no simple relationship was found between the expression level of EPO cDNA and the structure of the introduced expression vectors.     

Display of a Maize cDNA library on baculovirus infected insect cells

Background  

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings.