拟南芥信号肽AtRALF1的表达、纯化及活性分析

利用RT-PCR扩增获得拟南芥At RALF1基因的全长cDNA序列,将其构建入携带His标签的原核表达载体p ET28b中,获得重组表达载体p ET28b-At RALF1,并将其转入到大肠杆菌BL21(DE3)中。随后在不同的IPTG浓度、温度和诱导时间条件下,进行At RALF1蛋白的表达研究,建立了At RALF1融合蛋白的高效表达体系。结果表明,在30℃、1 mmol/L IPTG的条件下诱导4 h,At RALF1融合蛋白的表达量最大。进一步用获得的At RALF1融合蛋白处理苗龄5 d的野生型拟南芥(Col-0)植株,发现其根的生长受到了抑制,表明我们获得了具有活性的At RALF1小肽,为进一步研究该小肽奠定了基础。     

人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达

在原核系统中高效表达抗狂犬病毒单链抗体scFv41,以便进一步研究其生物学功能,预测临床应用前景。以重组质粒pCANTABscFv41为模板,PCR扩增带NcoI和NotI位点的scFv41基因,克隆入原核表达载体pET-22b( ),酶切鉴定重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。竞争ELISA检测表达蛋白的特异结合活性。酶切鉴定证实scFv41基因已插入原核表达载体pETl-22b( ),重组表达质粒pET-scFv41在大肠杆菌BL21(DE3)中获得了高效表达,表达量约占菌体蛋白总量的30％。竞争ELISA检测结果表明scFv41表达蛋白可特异抑制抗狂犬病毒IGY与狂犬病毒的特异性结合。该实验为进一步研究scFv41的生物学特性和免疫保护作用,及基因工程抗体的制备奠定了基础。     

拟南芥海藻糖酶基因克隆及其在大肠杆菌中高效表达与功能研究

从拟南芥幼苗中提取RNA,通过RT-PCR克隆得到海藻糖酶基因后,将其构建到原核高效表达载体pET30a( )上并在大肠杆菌BL21菌株中进行高效诱导表达,继而对纯化得到的海藻糖酶蛋白进行活性检测和酶学特性研究.实验结果表明,植物源的海藻糖酶基因在异体大肠杆菌中能够高效表达,纯化获得的海藻糖酶蛋白在试管条件下具有较高的海藻糖水解活性,其活性最适温度为45℃.通过GC-MS分离检测,可以明显地看到酶反应过程中底物海藻糖和产物葡萄糖的含量随反应时间变化的消长关系,这充分证明克隆基因在大肠杆菌中的表达产物具有海藻糖酶的功能.     

棉花GhVHA A基因的原核表达及其重组大肠杆菌的抗逆性分析

为进一步验证棉花GhVHA-A基因的功能,该研究将棉花GhVHA-A基因构建到原核表达载体pET28a上,利用IPTG诱导其在大肠杆菌BL21(DE3)中高效表达,同时对重组大肠杆菌BL21(pET28a-GhVHA-A)进行抗逆性分析。结果表明:(1)半定量RT-PCR分析发现,棉花幼苗液泡膜H+-ATPase基因(GhVHA-A)表达水平受脱水和高盐胁迫诱导。(2)将1 872bp长的编码区序列连接至原核表达载体pET28a上,成功构建了原核表达载体pET28a-GhVHA-A;SDS-PAGE电泳检测结果表明,在70kD左右处有1条特异表达的蛋白质条带,与预期的目的产物大小一致。(3)重组菌BL21(pET28a-GhVHA-A)的抗逆性分析发现,重组菌对PEG6000(20%)和NaCl(0.5mol/L)的抗性明显高于对照菌株BL21(pET28a),表明GhVHA-A基因在大肠杆菌中表达后能够增强菌株的抗性。本研究结果为GhVHA-A基因在植物抗逆基因工程中的应用提供了理论依据。     

血管生成抑制因子arresten基因的克隆表达

构建血管生成抑制因子arresten基因的原核表达重组体 ,并进行初步表达。从人胎盘组织中提取总RNA ,经反转录 聚合酶链式反应 (RT PCR)扩增出arresten基因 ;采用T A克隆法 ,将arresten基因克隆入pGEM T载体中 ,经DNA测序确认后 ,构建原核表达重组体pRSET Arr,转化大肠杆菌BL2 1 (DE3) ,用IPTG诱导表达。所获得的arresten基因经测序正确 ,并表达重组蛋白 ,经SDS PAGE分析 ,相对分子量为 2 6ku。构建的原核表达重组体pRSET Arr能高效表达重组arresten蛋白。     

拟南芥水杨酸受体表达与在体转录水平的研究

目的:从拟南芥叶中克隆水杨酸结合蛋白(SA binding protein 2,SABP2,也称水杨酸受体)基因sabp2进行异源表达并测定其活性.方法:从拟南芥叶RNA中通过反转录PCR扩增sabp2,将PCR产物克隆至载体pMD - 19T simple中,经测序验证后,再基于pET28a构建重组表达载体,转化至大肠杆菌BL21( DE3)并表达,检测重组蛋白的活性.另一方面,对sabp2在拟南芥中转录水平进行了研究.结果:PCR获得792bp的sabp2基因,并成功构建异源表达载体pET28a - sabp2.优化结果表明,在0.4mmol/L IPTG诱导下20℃培养8h,表达产物活性较强,具天然SABP2的特征性酯酶活性.该基因在拟南芥叶中转录模式呈SA应激性和组织特异性.结论:sabp2成功表达,不仅为筛选SA受体拮抗剂提供新的原核体系,而且为探讨SA与SABP2相互作用在植物防御过程中时空变化奠定基础.     

乌桕SsSAD基因的原核表达与纯化研究

乌桕是一种重要的木本油料树种。SAD(stearoyl-acyl ACP desaturase)是油料植物中将饱和脂肪酸转变成不饱和脂肪酸的一种关键脱氢酶。为了进一步揭示乌桕SsSAD的功能,该研究在大肠杆菌中表达了该蛋白。结果表明:(1)通过RT-PCR的方法从乌桕种子中克隆出了SsSAD基因编码区全长序列,并将其克隆到低温诱导的原核表达载体pCold TF上,构建原核重组表达载体pCold TF/SsSAD,转化大肠杆菌BL 21star(DE3)并获得原核表达工程菌株。(2)通过IPTG法低温诱导表达融合蛋白。该重组质粒在大肠杆菌中得到了高效表达,融合蛋白分子质量约为101kD,且在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化和Western blotting检测证实获得了重组蛋白,上述结果为进一步研究乌桕SsSAD的结构和功能奠定了基础。     

甘蓝型油菜BnCOP1基因的原核表达和纯化

COP1蛋白是植物光信号调节网络中介导光信号转导的一个关键因子,对植物生长发育起重要调控作用.通过RT-PCR方法从甘蓝型油菜中克隆BnCOP1基因编码区全长序列,并将其连接到冷诱导原核表达载体pCold TF上,构建原核重组表达载体pCold TF/BnCOP1,转化大肠杆菌BL(21)star.通过IPTG法低温诱导表达融合蛋白.结果表明,重组质粒在大肠杆菌中获得高效表达,融合蛋白分子质量约为128 kD,融合蛋白在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化和Western blotting检测证实获得了高纯度的重组蛋白,这些结果为进一步研究油菜BnCOP1的结构和功能奠定了基础.     

SDH全基因的拼接及原核表达

目的:构建闭联异松树脂醇二脂脱氢酶(SDH)全基因片段与原核表达质粒载体,在大肠杆菌中进行表达.方法:将SDH3'端和5'端序列通过PCR方法拼接获得全基因后,然后将其插入到相应的原核表达质粒载体PGEX-6p-1中,形成了重组载体,并使其在大肠杆菌BL21中经IFFG诱导其表达蛋白,再用SDS-PAGE电泳检测表达结果.结果:成功的将SDH基因片段拼接成全基因,并将其构建入原核表达载体PGEX-6p-1中.测序结果表明载体构建成功,无插入和移码突变.经1.5mmol/LIPTC诱导后获得与预测大小(29.253kDa)完全一致的目的蛋白即闭联异松树脂醇二脂脱氢酶蛋白,并测得在37℃、250r/min的实验条件下的最佳诱导时间为4h.结论:成功获得了重组质粒并在大肠杆菌中较好的诱导表达得到了闭联异松树脂醇二脂脱氢酶的蛋白.     

雪莲类PEBP基因在大肠杆菌中的表达及鉴定

本实验旨在构建雪莲类PEBP基因原核表达载体,在大肠杆菌中表达并纯化类PEBP基因所编码的蛋白,为进一步研究奠定基础.将雪莲类PEBP基因开放阅读框序列克隆到原核表达载体pET30( )上,转化感受态表达菌株BL21(DE3),低浓度IPTG低温诱导融合蛋白的表达,纯化产物,Western blotting鉴定目的蛋白.IFTG低诱导PEBP,经SDS-PAGE分析,其相对分子量约为28 kD,与预期相符,表达量约占菌体蛋白的26.8%,并且通过亲和层析纯化了重组融合蛋白,Western blotting鉴定为阳性.成功构建了原核表达载体pET-PEBP,获得了高效表达产物,并为进一步研究雪莲类PEBP基因的抗冻功能打下基础.     

High-level expression of full-length antibodies using trans-complementing expression vectors

Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor-intensive cloning and screening steps. We describe a mammalian expression system for the high-level production of full-length antibody molecules. It has been shown that the dihydrofolate reductase (DHFR) selectable marker can be divided into two fragments that, with the aid of a leucine zipper, can re-associate to form an active molecule. Using bicistronic vectors, we linked the expression of each antibody chain to the expression of a DHFR fragment. Survival in selective media requires expression of both DHFR fragments that, by virtue of these vectors, also selects for the expression of both antibody chains. Initial pools produced 5 microg of Ab/10(6) cells/d (qP = microg/10(6) cells/d). Expression of each antibody chain in conjunction with a portion of DHFR also leads to concurrent amplification of both antibody chains in the presence of methotrexate, a DHFR inhibitor, and results in a two- to fivefold increase in antibody production with basal qPs ranging from 10-25 ug/10(6) cells/d. Shake-flask cultures of amplified pools produced up to 600 mg/L of antibody in 7 days. This system allows for rapid generation of antibodies without cloning and greatly simplifies selection of cell lines for the production of potential antibody therapeutics.     

Normalization and quantification of differential expression in gene expression microarrays

Array-based gene expression studies frequently serve to identify genes that are expressed differently under two or more conditions. The actual analysis of the data, however, may be hampered by a number of technical and statistical problems. Possible remedies on the level of computational analysis lie in appropriate preprocessing steps, proper normalization of the data and application of statistical testing procedures in the derivation of differentially expressed genes. This review summarizes methods that are available for these purposes and provides a brief overview of the available software tools.     

高效可溶性重组蛋白表达载体的构建

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。     

Tumor-specific gene expression patterns with gene expression profiles

Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, “RFE_Relief algorithm” was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.     

Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector

The baculovirus expression vector system (BEVS) has been widely used for over-expressing eukaryotic proteins due to a close resemblance in post-translational modification, processing, and transportation properties of the expressed protein, to that of the mammalian cells. In comparison to the bacterial expression system, protein yield from BEVS is relatively low, resulting in higher cost of production. To improve the existing recombinant protein expression levels, baculovirus homologous region1 (hr1) was strategically integrated into the bacmid-based transfer vectors. Luciferase reporter, human Protein Kinase B-α (PKB-A), and N-terminal-modified CYP-1A2 genes were independently cloned in non-hr1 and hr1 constructs for generating respective bacmids and baculoviruses. These recombinant baculoviruses were utilized for comparing the expresion levels at varying multiplicity of infections (MOI) and time intervals in Spodoptera frugiperda (Sf21) or Trichoplusia ni (Tni) insect cell lines. Targeted insertion of hr1 upstream to CYP-1A2, PKB-A, and Luciferase genes, compared to the non-hr1 sets, led to 3-, 3.5-, and 4.5-fold increase in the resultant protein levels, respectively. Moreover, at equal protein concentration, the corresponding activity and inhibition characteristics of these high expression hr1 sets were comparable to that of the respective non-hr1 sets. Utilization of this modified baculovirus expression construct offers significant advantage of producing recombinant proteins in a cost-effective manner for various biotechnological and therapeutic applications.     

Tumor-specific gene expression patterns with gene expression profiles

Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.     

Creation of genome-wide protein expression libraries using random activation of gene expression

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.