重组人红细胞生成素二聚体不同真核表达载体的构建及表达

为构建重组人红细胞生成素(recombinant human erythropoietin,rhEPO)二聚体真核表达载体,应用PCR方法扩增EPO cDNA,PCR产物克隆入T载体后,经酶切、连接、转化等过程分别构建了3个EPO二聚体的真核表达载体pBT-1c、pBT-2s及pBT-3c,经测序序列完全正确。然后将3个真核表达载体分别转染于CCS-7细胞及CHO-dhfr-细胞中,用ELISA方法检测,它们在COS-7的瞬时表达量分别为4IU／ml、11.5IU／ml和7.2IU／ml。其中EPO二聚体真核表达载体pBTsv稳定转染CHO-dhfr-细胞后,用氨甲喋呤(MTX)逐渐加压的方法筛选到阳性克隆,表达量可达到4000IU／106cells／72h。     

人工转录因子促进外源基因在CHO细胞中的高效表达

以酵母转录因子GAL4中1-147位氨基酸序列为构建人工转录因子的DNA结合结构域,单纯疱疹病毒转录激活子VP16中12肽(DALDDFDLDMLG)的4个串联重复作为人工转录因子的功能结构域,用SV40的核定位序列(NLS)将两部分连接起来,构建了人工转录因子GVP4并将其克隆进入表达载体pcDNA3·1/Hygro( )中。将不同长度的人工转录因子结合序列构建在外源基因表达载体pcDNA3·1( )启动子CMV的上游,分别连接外源基因EGFP和tPA。用表达人工转录因子和外源基因的载体共转染CHO细胞,EGFP和tPA在含不同数量人工转录因子结合位点表达载体pcDNA3·1( )转染的CHO细胞中的表达水平呈现不同程度的提高。其中,以引入10个人工转录因子结合位点的表达载体的效果最明显,EGFP和t-PA的表达效率均提高2~3倍。结果表明,人工转录因子能有效地促进外源基因在哺乳动物细胞中的表达。     

大鼠促甲状腺激素释放激素受体1基因的克隆及其在COS-7细胞系中的表达

目的:克隆大鼠促甲状腺激素释放激素受体1(TRH-R1)基因,构建其真核表达载体,并检测该基因在非洲绿猴肾细胞系COS-7中的表达。方法:应用RT-PCR方法,以大鼠脑源RNA为模板,扩增获得TRH-R1基因,定向克隆到pDsRed2-N1中,以LipofectAMINE 2000试剂转染pDsRed2-N1-TRH-R1表达载体至COS-7细胞系中进行瞬时表达。结果:测序结果表明,从大鼠脑源总RNA中克隆到正确的TRH-R1基因全长编码序列;显微照相观察到所构建的TRH-R1表达载体质粒在COS-7细胞系中获得有效表达。结论:大鼠TRH-R1基因的克隆、真核表达载体的构建及在COS-7细胞系中表达获得成功,为进一步研究其功能奠定了基础。     

通用真核质粒表达载体的构建

利用真核基因表达调控的原理,以pSV2-dhfr为起始材料,构建了两个通用的真核质粒表达载体pMAML1-dhfr和pMAML2-dhfr,它们包含人巨细胞病毒立即早期启动子/增强子调控元件,由两个转录方向相同或相反的表达单元组成.以荧火虫荧光素酶基因为报道基因,β-半乳苷酶基因为内对照,借助COS-7短暂表达系统,研究了它们对荧光素酶表达的影响,并比较了它们与出发载体pSV2-dhfr的相对强弱.     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因（k2tPA）、扩增基因（dhfr）、重组酶识别序列（FRT）及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO／dhfr^-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hotspot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO／dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17．1μg／10^6cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

结核分枝杆菌furA基因真核表达质粒的构建及表达

以BamH Ⅰ和Hind Ⅲ双酶切pRSET-furA,获得结核分枝杆苗铁调控基因furA,将其克隆入真核表达载体pcDNA3．1（-）,构建了重组质粒pcDNA-furA,经酶切鉴定正确后,将重组质粒以阳离子聚合物转染CHO细胞。经RT—PCR分析表明,furA可在CHO细胞中转录;用间接免疫荧光检测,表达有FurA蛋白的细胞着染。以上结果表明,通过构建结核分枝杆菌furA基因的真核表达载体pcDNA-furA,使知以基因可以在CHO细胞中表达。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k₂tPA)、扩增基因（dhfr）、重组酶识别序列(FRT)及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr^－细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hot spot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hot spot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k₂tPA的高表达,表达量达到17.1μg/10⁶cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

小鼠pEGFP-Hoxa11真核表达载体的构建及表达

目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.     

分泌表达bFGF的中国仓鼠卵巢(CHO)细胞的构建及其特性分析

CHO细胞在无血清或无蛋白培养条件下培养通常会遇到贴壁能力差,细胞活力差等问题。通过构建分泌型bFGF基因,克隆到pIRESneo3表达载体上,转染CHO细胞,通过MTT法间接检测细胞培养上清中bFGF表达,并在无蛋白培养基中观察细胞的生长。结果显示转染的CHO细胞表达bFGF,且分泌的bFGF有生物活性;转染的CHO细胞在无蛋白培养基中较未转染的CHO细胞的贴壁能力和活力强。成功改造了CHO细胞,为CHO细胞在无血清或无蛋白条件下大规模培养提供了基础。     

人转化生长因子β1 cDNA在COS－7细胞中的表达

在测定了ＴＧＦβ１全长序列的基础上,构建了两个ＴＧＦβ１的表达载体ｐＣＭＶβ和ｐＳＶＬβ;分别从转录水平和翻译水平检测了这两个载体在ＣＯＳ－７细胞中的表达。同时利用ＣＯＳ－７短暂表达系统,建立了ＴＧＦβ１的生物测活法;并分别研究了上清收获时间、表达载体、转染方法对ＴＧＦβ１表达的影响     

Enhanced expression of transgene in CHO cells using matrix attachment region

The expression of transgenes in mammalian cells is often at a low level mainly due to position effects from the neighboring chromatin context. To improve this, we have constructed a vector pCAM, which contains chloramphenicol acetyltransferase (CAT) reporter gene cassettes, driven by SV40 early promoter and flanked by two human beta-globin MARs in cis. We transfected this vector into the Chinese hamster ovary (CHO) cell line, and found that the level of CAT gene expression with MAR was effectively increased, about 5.493-fold higher than those without MARs. Moreover, the variations of CAT expression among individuals of transformants were decreased 2.670-fold. Our result also showed that MAR could increase the proportion of positive colonies in recombinants.     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF‐1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT‐PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.     

人重组白细胞介素12在CHO细胞中的表达

将人白细胞介素12（interleukin 12, IL-12）两条链p35及p40全长cDNA分别亚克隆至真核表达载体pcDNA3中,构建了pcDNA3/p35a,pcDNA3/p40a,pcDNA3/p35b,pcDNA3/p40b四种真核细胞重组表达质粒,利用磷酸钙共沉淀法转染中国苍鼠卵巢(CHO)细胞. 通过对阳性克隆的筛选鉴定,获得了稳定表达人IL-12的CHO细胞株,活性最高的一株表达量为105 U/ml.此细胞株经半年的传代培养,能够稳定地分泌IL-12.结果显示：IL-12在CHO细胞中的稳定表达受到重组质粒结构、DNA整合、mRNA转录、蛋白质翻译等多因素的影响,IL-12两个亚基在CHO细胞中的共同表达是产生有生物活性IL-12的基础.     

SOCS-3过表达对红系基因表达的影响

目的:建立能稳定、高效表达细胞因子信号转导抑制因子-3(SOCS-3)的细胞株SOCS-3-K562,为探讨SOCS-3在造血发育中的作用奠定基础。方法:通过重组慢病毒系统感染人红白血病细胞K562,采用流式细胞分选术,根据绿色荧光蛋白表达情况,获得稳定高表达SOCS-3的K562细胞;利用实时荧光定量PCR和蛋白质印迹实验,比较分选获得的细胞与对照细胞的SOCS-3表达差异;利用半定量PCR检测SOCS-3表达升高对K562红系发育相关基因GATA-1、β-globin表达水平的影响。结果:构建了人SOCS-3慢病毒表达载体;与对照组相比,通过流式细胞分选获得的K562细胞的SOCS-3基因表达水平升高8.05倍,蛋白表达水平升高3倍;SOCS-3表达升高后,K562细胞的GATA-1、β-globin基因表达受到明显抑制。结论:SOCS-3在造血发育中有重要的调控作用,而对其表达进行改变将在规模化的造血细胞定向诱导研究中发挥重要作用。     

New adenovirus vectors for protein production and gene transfer

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression. This revised version was published online in August 2006 with corrections to the Cover Date.