重组pEGFP—C2-p100-TSN点突变质粒构建及表达

目的将6个不同的p100-TSN．Mutants基因片段分别定向连入PEGFP—C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础。方法利用EcoR I和Xho I双酶切方法从6个pcDNA3．1（＋）-p100-TSN．Mutants重组质粒中分别获得p100-TSN．Mutants的cDNA片段,将其连人pEGFP—C2质粒载体中,再将成功构建的6个pEGFP—C2-p100-TSN．Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达。结果①将重组质粒进行双酶切鉴定可见p100-TSN．Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达。结论①6个pEGFP—C2-p100-TSN．Mutants重组质粒构建成功;②p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达。     

沙眼衣原体真核表达质粒pcDNA4/CT703的构建及其表达

目的：构建含沙眼衣原体（Chlamydia trachomatis, Ct）基因CT703的真核重组表达质粒pcDNA4/CT703,并检测其在HeLa细胞中的表达.方法：利用RT-PCR扩增CT703基因,然后将其亚克隆到真核表达载体pcDNA4,PCR、双酶切和测序检测重组质粒.将正确的重组质粒瞬时转染HeLa细胞,免疫荧光和Western Blot实验检测重组质粒目的蛋白表达. 结果：经PCR、双酶切和测序鉴定后,成功构建了真核重组表达质粒pcDNA4/CT703,将其转染HeLa细胞后,免疫荧光和Western Blot实验能检测到目的蛋白的表达.结论：成功构建了重组质粒pcDNA4/CT703,并能在HeLa细胞中表达,为进一步研究CT703的功能奠定了基础.     

pAd—SIG腺病毒质粒构建及其在MCF-7细胞中的表达

目的含人生长抑素受体2亚型（ human somatostatin receptor subtype 2, hSSTr2 ）和增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）的腺病毒重组质粒的构建及表达。方法采用PCR方法将hSSTr2以及IRES-EGFP基因亚克隆至穿梭质粒pShuttle—CMV中,获得pShuttle-CMV／hSSTr2-EGFP,经PmeI酶切,去磷酸化后转化含pAdeasy-1的超感受态BJ5183菌,细菌内同源重组构建腺病毒质粒pAdeasy-1／hSSTr2．EGFP（pAd．SIG）,将其转染HEK293细胞进行病毒包装与滴度测定。重组腺病毒感染MCF-7细胞,流式细胞术检测其表达。结果酶切、PCR及基因测序证实重组腺病毒质粒pAd-SIG构建成功,病毒滴度为8．2×10^9 IU／ml,流式细胞术结果显示hSSTr2在MCF-7细胞中表达阳性率为86．59％。结论成功构建了区组腺病毒质粒pAd-SIG,并能在真核细胞表达,为体内外研究hSSTr2效应提供了基础。     

重组大肠杆菌碱裂解方法的改进

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。     

口蹄疫病毒多基因重组质粒的体外表达及衣壳组装

PCR扩增获得包含口蹄疫病毒P1、2A、3C、3D及部分2B编码区的目的基因片段P12X3C3D,将P12X3C3D经AflⅡ和XbaⅠ双酶切后,定向克隆于真核表达质粒载体pcDNA3.1( );另将PCR扩增获得的P12X3C3D直接与真核表达质粒载体pTARGET^TM连接,进行筛选、鉴定及DNA序列分析,分别获得重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D。将重组质粒分别转染BHK-21细胞,通过双抗体夹心ELISA方法和间接免疫荧光标记方法检测细胞中表达的口蹄疫病毒抗原,用磷钨酸负染,以电子显微镜观察转染重组质粒的细胞中组装的口蹄疫病毒空衣壳。结果表明,口蹄疫病毒基因组片段正确克隆到真核表达质粒载体上,重组质粒pcDNA3．1／P12X3C3D、pTARGET／P12X3C3D均可在BHK-21细胞中表达FMDV目的蛋白。其中重组质粒pTARGET／P12X3C3D表达的口蹄疫病毒抗原蛋白,能够在细胞内正确组装成病毒空衣壳。     

Ad-hBNP重组腺病毒的构建

目的:构建携带人BNP cDNA片段高效重组腺病毒Ad-hBNP,为实验提供研究工具.方法:从人心肌组织提取的RNA,用RT-PCR方法中获得hBNP扩增片段,与pUCm-T载体连接构成pUC-hBNP重组质粒;用KpnI、SalI分别双酶切pUC-hBNP和pAd-Track-CMV,将目的片段插入pAd-Track-CMV构建重组质粒pAdTrack-CMV-hBNP;pAdTrack-CMV-hBNP重组质粒经Pmcl酶切线性化后,电转法转入含有腺病毒骨架质粒的E.coli BJ5183感受态细胞中,同源重组获得重组质粒pAdEasy-hBNP;经BamHI、PacI酶切鉴定及基因序列检测后,重组成功的pAdEasy-hBNP经阳离子脂质体法转染HEK293T细胞,经过包装、扩增和纯化后,测定病毒滴度,电镜检测病毒形态.结果:转染HEK293T细胞5-6天GFP呈"彗星"状;重组腺病毒滴度为1.1×1012V.P/ml;电镜检测重组腺病毒为多面体结构.结论:应用Ad-Easy缺陷性腺病毒载体系统成功构建重组腺病毒Ad-hBNP,为进一步基因治疗研究提供分子生物学工具.     

促DNA修复多效蛋白诱导因子pprI逆转录病毒载体系统的构建及其鉴定

目的:为实现耐辐射球菌pprI基因在哺乳动物细胞中的稳定遗传与表达,构建重组逆转录病毒载体质粒pLXIN-pprI。方法:将目的基因pprI亚克隆经过双酶切后定向连接到pLXIN质粒上,构建逆转录病毒重组质粒pLXIN-pprI。将pLXIN-pprI转化大肠杆菌感受态细胞DH5α,氨苄青霉素筛选后抽提获得重组质粒pLXIN-pprI,双酶切及DNA测序鉴定。结果:酶切鉴定及测序结果显示结果与预期相符,pprI基因成功插入pLXIN逆转录病毒载体中。结论:重组逆转录病毒载体质粒pLXIN-pprI构建成功,为实现耐辐射球菌pprI基因在哺乳动物细胞中的重组与表达奠定了基础。     

STAT3小干扰RNA的构建及对缺氧复氧人肾小管上皮细胞凋亡的影响

目的:构建信号转导与转录因子3(STAT3)小干扰RNA(siRNA)表达载体,并观察其对缺氧复氧后人肾小管上皮细胞(HKC)凋亡的影响。方法:设计3对人STAT3 siRNA靶序列,用DNA重组技术克隆至质粒pRNAT-U6.1/neo中,构建重组质粒pRNAT-U6.1-STAT3 siRNA,检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞,Western blotting和Real Time-PCR测定STAT3蛋白和mRNA表达量,流式细胞仪测定细胞凋亡,间接荧光法测定Bcl-2和Bax表达的变化。结果:靶向STAT3基因表达的质粒载体构建成功,并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC细胞STAT3表达、凋亡率和Bax/Bcl-2比值增加;缺氧复氧后HKC细胞转染重组质粒后STAT3表达、凋亡率和Bax/Bcl-2比值明显降低。结论:成功构建并筛选最佳抑制效率的靶向STAT3的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3信号转导通路的活化,并进一步通过上调Bcl-2、下调Bax蛋白的表达,从而抑制细胞凋亡。     

CDADC1在人视网膜色素上皮细胞中功能研究

目的：研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法：采用基因重组技术构建荧光表达载体pEGFP－C1－CDADC1和真核表达载体pcDNA3．1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP—CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果：FP—CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD—NA3．1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19．37％,pcDNA3．1-myc空载质粒转染组S期细胞占10．87％,而空白对照组S期细胞占3．33％,重组质粒转染组与两对照组之间的差异有统计学意义（P〈0．01）。结论：CDADC1在增生性玻璃体视网膜病变（PVR）发生和发展过程中可促进DNA的合成,引起细胞增生。     

酿酒酵母超氧物歧化酶(SOD)基因的克隆和表达

通过PCR扩增技术从酿酒酵母中得到了Cu,zn—SOD的结构基因,此基因被亚克隆到大肠杆菌质粒载体pT7—7．得到重组质粒pT7－7：SOD。利用EcoRI和Pstl酶切pT7-7::SOD质粒．经琼脂糖凝腔电泳,DEAE-滤膜回收Cu。zn—SOD结构基因片段,将其亚克隆到M13中．并转化大肠杆菌,得到了重组质粒M13-::t SOD,酶切和纯化后的SOD基因,定向克隆到酵母质粒载体pHz－8的smal和EcoRI位点上,构建成重组质粒pHZ－8－l。经转化酵母受体菌ZH-l和DP—l后得到了转化子．来自于ZH—l的转化子在非选择性条件下培养40世代后仍有95％以上细胞保留重组质粒。而来自于DP-1的转化子很不稳定。经蛋白提取、聚丙烯酰胺凝胶电泳和酶活性测定结果表明,来自于zH－1转化子中SOD的表达量约为细胞可溶性蛋白的15％．并具有生物活性。     

Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.     

快速筛选高效表达重组蛋白毕赤酵母菌株新方法的建立及评价

毕赤酵母是当前应用最为广泛的重组蛋白表达系统之一,文中建立了一种快速筛选高效表达重组蛋白的毕赤酵母菌株的新方法。首先,对内质网转膜蛋白Sec63融合表达增强型绿色荧光蛋白EGFP的改造菌株GS115-E表达重组蛋白的能力进行检测;之后将携带不同拷贝数的植酸酶phy基因或木聚糖酶xyn基因的质粒转化进入GS115-E中,得到具有不同植酸酶或木聚糖酶表达水平的重组菌株,分别检测不同菌株的EGFP与重组蛋白的表达水平;最后,利用分选型流式细胞仪,根据绿色荧光值的高低对包含不同植酸酶表达水平的重组菌株的菌群进行分选。结果显示重组菌株中EGFP的荧光值与重组蛋白的活性表达水平之间具有良好的线性相关性(0.8|R|1),且利用流式细胞仪可高效地从混合菌群中筛选得到高产菌株,所分选得到的高荧光菌株在摇瓶发酵120 h时植酸酶表达水平是低荧光菌株的4.09倍。本方法通过检测菌株的EGFP荧光值代替检测重组蛋白的表达水平和活性,从而实现高表达菌株的筛选,大大提高了其应用的便捷性及通用性。与流式细胞仪、液滴微流控等高通量筛选仪器或技术结合将进一步提高筛选的速度与通量,为筛选获得高效表达重组蛋白的毕赤酵母菌株提供了简便、快速的新途径。     

Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation

The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.     

Expression and purification of recombinant nattokinase in <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> cells

A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.     

Electrophysical monitoring of culture process of recombinant Escherichia coli strains

A novel method for monitoring the cell culture process has been developed. The method is based on the measurements of electro-optical characteristics of cell suspension, calculation of cell structure parameters, and the relationship between accumulation of proteins and change of these parameters' employment. Application of the method for the monitoring of a culture process of a recombinant strain is considered. The process of growth of recombinant strains cannot be sufficiently predicted and the direct measurement of cell culture parameters is unlikely to be the most efficient way of solving the problem.Escherichia coli plasmid-free and recombinant strains synthesizing the fusion protein consisting of tumor necrosis factor-alpha (TNF) and thymosin-alpha(1) (T) were studied. It was found that cytoplasmic electroconductivity of the strains investigated increased during the culture process. The accumulation of insoluble recombinant pThy-315-encoded hybrid protein TNF(SINGLEBOND)T in cells resulted in a decrease of the membrane dielectric permeability. To determine variations of membrane dielectric permeability the amount of insoluble recombinant protein TNF(SINGLEBOND)T in the bacterial cells should be calculated.     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

重组人E1A激活基因阻遏子/myc-His融合糖蛋白的纯化及功能鉴定

目的:纯化重组人E1A激活基因阻遏子(hCREG)糖蛋白,验证重组hCREG糖蛋白具有抑制体外培养的人胸廓内动脉平滑肌细胞(HITASY)增殖的生物学功能。方法:根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。用流式细胞周期分析研究添加0.5μg/ml、1μg/ml及2μg/ml重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究重组蛋白对体外培养HITASY细胞增殖的影响。结果:根据6×His亲和层析原理用Ni-NTA纯化重组hCREG蛋白,浓缩并脱盐后的重组hCREG蛋白浓度为1.6mg/ml,纯度为92%,此蛋白仍保留了糖基化形式。流式细胞仪细胞周期分析结果提示重组hCREG蛋白可抑制体外培养的HITASY细胞增殖,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg/ml重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,组间比较有统计学差异(P0.05)。结论:具有生物学功能的重组hCREG/myc-His糖蛋白的成功纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。     

Immobilization of mouse single-chain antibodies for affinity chromatography using the cellulose-binding protein

A laboratory method for obtaining immunoaffinity medium for chromatographic purification of recombinant human interferon alpha2b (IFN-alpha2b) is described. The method is based on oriented and non-covalent immobilization of recombinant antibody fragments on cellulose. The single-chain fragment variable (ScFv) against human IFN-alpha2b was genetically fused to cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome and expressed in Escherichia coli. After the isolation of the target protein in functionally active form from bacteria cells its bioaffinity immobilization on several forms of cellulose powders has been carried out. The crystalline microgranular cellulose with immobilized ScFv-CBD-fusion protein was used as affinity medium to perform the purification of recombinant human IFN-alpha2b directly from clarified extract of E. coli cells.     

Endoplasmic reticulum‐directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide‐dependent manner, to acquire specific post‐translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER‐directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER‐directed mRNA, with no cytoplasmic translation of ER‐directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER‐directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.