argE基因在大肠杆菌中的表达优化

N-乙酰鸟氨酸脱酰基酶可在重组菌BL21（DE3）-pET22b-argE中表达。首先确定了该酶的细胞表达定位,再研究了诱导温度、诱导剂种类及浓度、诱导起始菌体密度、诱导时间等因素对重组菌生长及目的蛋白表达活性的影响。结果表明,IPTG和乳糖皆可诱导目的蛋白表达,乳糖的诱导效果优于IPTG。在诱导起始0D600为0．46时加入15g／L乳糖,20℃诱导18h最适于目的蛋白的活性表达。表达条件优化后,酶活从1．68U／mL提高至282．99U／mL,约为原来的168倍。     

虹鳟肿瘤坏死因子(TNFα)基因体外表达与纯化的研究

将虹鳟两种肿瘤坏死因子TNFα和TNFα2基因的成熟肽编码区域,用带有BamHI和HindⅢ酶切位点的基因特异性引物进行：PCR扩增。扩增片段用限制性内切酶消化并连接到pQE30表达载体上,连接产物转化到大肠杆菌JM109感受态细菌中。转化子经PCR筛选,质粒测序,完成了虹鳟两种TNFα基因重组子的构建。重组子经体外培养和诱导后,获得了高效表达的TNFα重组蛋白。高效表达的重组TNFα不受诱导剂IPTG的影响,并且由于重组子高效表达而形成了包涵体;重组蛋白产量约占菌体蛋白总量的25％—30％。应用Ni-NTA和固定金属亲和层析(IM-PC)技术,在变性条件下获得了高度纯化的重组蛋白,纯化重组蛋白的产量约为0．5—1mg／L。     

用E．coli表达Canstatin—N及其表达条件优化

以重组质粒DET—CN为模板设计引物CASN1N和CASN2,PCR方法扩增约267bp的人血管能抑素N端1～89氨基酸基因片段,用EcoRI和Sal I双酶切将其克隆进pET-22b（＋）载体获得重组表达质粒pET-22b（＋）一CN,转化E．coliBL21（DE3）,用IPTG诱导表达Canstatin-N,产物以包涵体形式存在。本文在摇瓶发酵条件下研究了诱导剂浓度、诱导培养时间对目标蛋白表达的影响,结果表明IPTG的最佳诱导浓度为0．1mmol／L;37℃下诱导培养2h时产物表达量最高。纯化获得的融合hiS6的重组Canstatin—N具有免疫和抑制鸡胚绒毛尿囊膜（CAM）新生血管生成活性。     

志贺毒素B（ShT-B）亚单位在两种表达载体中表达形式分析

用KpnⅠ和HindⅢ双酶切pGEMTB3克隆质粒,得到大小约为225bp的ShTB基因片段,分别将其插入到经双酶切的pQE40和pQE30表达载体中,构建了2个ShTB的重组表达质粒pQE40B3和pQE30B2,分别转化到E.coliM15,经IPTG诱导后,重组质粒目的蛋白均得到表达。其中,pQE40B3表达蛋白约占菌体总蛋白的37%,主要为包涵体形式。pQE30B2表达蛋白约占菌体总蛋白的16%,主要为可溶性形式,约9.2%。为重组抗原的制备提供了必要的物质基础。     

采用正交设计优化HPV16L1重组蛋白表达的实验研究

通过正交试验对构建的人乳头瘤病毒（HPV）16型L1蛋白的重组表达菌株pQE31—HPV16L1／M15（pREP4）进行表达条件的优化,以增加目的蛋白的表达量。实验选取影响蛋白表达的4个主要因素,分别为诱导时间、诱导剂浓度、细菌密度和诱导温度,采用四因素两水平正交试验,通过SDS-PAGE及Western blot测定重组表达蛋白,以Tanon凝胶成像系统分析目的蛋白含量作为检测条件优化效果的指标,所得数据采用SAS软件进行统计分析。实验结果表明在37℃、IPTG0．5mmol／L,菌密度OD600为1．0的条件下诱导4h所得蛋白量最大;经统计正交分析的最佳诱导条件为37℃、0．5mmol／LIPTG和菌密度OD600为1．0的条件下诱导7h。经实验验证,应用正交分析的最佳条件诱导所得目的蛋白量最大,但实际中通常采用诱导4h条件。实验证明正交试验可以用于基因工程菌株蛋白表达条件优化过程,提高工作效率。     

荧光素酶基因luc在大肠杆菌中表达条件优化

对重组荧光素酶大肠杆菌菌株M15／pQE30-luc进行了表达条件的优化研究。单因素结果表明：在初始pH值7．0,装液量为20％,2％的接种量,终浓度为0．5mmol／L的IPTG,添加10—30mmol／L的Mg^2＋,摇床转速为200r／min,37℃诱导3．5h酶的表达量最高。正交试验结果表明：初始pH值为7．0,添加40mmol／LMg^2=,接种量2％,装液量为20％时表达量最高,比酶活达1．63×10^8RFU／mg蛋白。     

构建羰基还原酶基因工程菌生物转化产l-麻黄碱

以筛选得到的Morganella morganii J-8细菌的基因组为模板,通过PCR扩增得到目的基因mdlh2。核苷酸序列测定结果表明,基因全长1046bp。以pET28a（＋）为表达载体,构建重组质粒pET28a（＋）-mldh2,并在E．coli BL21（DE3）中表达。利用表达产物进行生物转化,发现其具有催化底物1-苯基-2-甲氨基丙酮（简称MAK）产l-麻黄碱的活力。进一步考察了诱导时间和IPTG浓度对重组菌表达的羰基还原酶的影响,37℃下用0．5mmoL／L的IPTG诱导4h,重组羰基还原酶的酶活达到0．2U／mg蛋白,转化液中l-麻黄碱质量浓度达到45mg／L。     

Pseudomonas putida S1海藻糖合成酶基因在大肠杆菌中的克隆表达

利用PCR和TA克隆方法扩增和克隆得到了恶臭假单胞菌Pseudomonas putida S1的海藻糖合成酶基因treS.对其进行序列分析表明,其编码区含有2067bp,编码含688个氨基酸残基的蛋白质,其核苷酸序列和蛋白质序列与来源于其它假单胞菌属细菌的海藻糖合成酶的序列表现出了较高同源性.将该基因序列与表达载体pQE30T连接,构建重组质粒pQE30T-TS,并将其转化至E.coli M15菌株中.重组菌株经诱导表达后SDS-聚丙烯酰胺凝胶电泳结果显示有明显的分子量约77.5kD的特异蛋白条带出现.经测定酶活力达19U/mL,约是原始菌株P.putida S1的50倍.     

转化生长因子α－绿脓杆菌外毒素融合蛋白TGFα－PE40高效表达质粒的构建和表达

利用基因工程技术,将质粒pYX382用xbaI和EcoRl切下插入的TGFa—PE40融合 基因片段-连接到可表达载体pCB604的XbaⅠ／EcoR Ⅰ位点中,构建成新的重组质粒p2x—TP1。P2x—TP1转化E.coli BL2l感受志菌后,在ⅠPTG诱导下Ⅰpp启动子转录表达TGFα—PE40融合蛋白。表达产物主要以包涵体形式沉积在细胞内。融合蛋白表达量的高低与诱导时的细胞密度,诱导的温度以及培养基有关。在一定范围内与诱导剂的剂量以及诱导时间的长短无关。P2x—TPl重组质粒在工程菌中的表达TGFα－PE40融合蛋白量约为50mg／L。     

乳糖诱导耐热木聚糖酶基因在大肠杆菌中表达

考察乳糖代替IPTG诱导耐热木聚糖酶基因在重组大肠杆菌BL21中表达的可行性。分别以IPTG和乳糖作为诱导剂,对诱导时机、诱导剂浓度、诱导持续时间等主要因素进行了分析比较。结果表明,当菌体生长至发酵液吸光值OD600达1．3时加入乳糖其终浓度达29．2 mmol／L,37℃,持续诱导9．5h,木聚糖酶活力达到最高,为3587.5U,是IPTG优化条件下持续诱导7h达到的最高酶活的2．07倍,而成本只有IPTG的1／204。     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli

The alanine transporter (alanine carrier protein, ACP) gene of thermophilic bacterium PS3 was previously cloned and expressed in a functionally active form in Escherichia coli cells. To achieve controlled overproduction of the ACP protein, we designed a plasmid encoding a fusion protein comprising ACP joined to the carboxyl terminus of the maltose binding protein (MBP-ACP). Upon transduction of the plasmid into E. coli RM1 cells defective in alanine/glycine transport, the transport activity was expressed even before induction with 1-thio-beta-D-galacto-pyranoside (IPTG), and increased slightly on induction with IPTG at low concentrations. However, overexpression of the MBP-ACP gene, induced by higher concentrations of IPTG, resulted in death of the host cells. Hence we screened other host cells and found that the MBP-ACP fusion protein was produced in a large quantity in E. coli TB1 cells 3 h after IPTG induction. The MBP-ACP fusion protein was accumulated in cytoplasmic membranes in an amount reaching more than 20% of the total membrane protein. The affinity-purified MBP-ACP exhibited very low transport activity when reconstituted into proteoliposomes.     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV－1囊膜基因gp85片段来克隆到表达质粒pET-21d( )中得到重组表达质粒pET-21d-RAV-1env(BelⅡ/SalⅠ）序列分析表明该插入片段的核苷酸序列和阅读框都与RAV－1囊膜基因相应序列相同。用其转化大肠杆菌BL21（DE）3并经IPTG诱导,SDS－PAGE分析表明RAV－1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱持续时间对表达量的影响更大。     

Enhanced expression of a recombinant malaria candidate vaccine in Escherichia coli by codon optimization

This study was conducted to compare the expression of three constructs of a multistage candidate vaccine (FALVAC-1) against Plasmodium falciparum in an Escherichia coli system: a synthetic gene with P. falciparum codons, a synthetic gene with optimized E. coli codons, and a synthetic gene with P. falciparum codons co-transformed with a RIG plasmid, which encodes three tRNAs (AG(A/G), ATA, GGA) that recognize rare E. coli codons. The expression of the protein increased at least threefold with codon optimization. The presence of the RIG plasmid in the co-transforming cells did not significantly increase the expression level of the gene with P. falciparum codons. The growth of cells transformed by the construct with P. falciparum codons was significantly slower than that of cells transformed by the construct with optimized E. coli codons after induction of protein expression with IPTG. The cells containing the non-codon optimized gene co-expressed with RIG plasmid had the slowest growth at all time points in culture. Thus, codon optimization significantly increases the yield of P. falciparum candidate vaccines in the E. coli expression system.     

鼻咽癌侯选抑瘤基因BRD7原核表达载体的构建及其表达

BRD7基因是一个鼻咽癌侯选抑瘤基因,为了构建BRD7基因的原核表达载体并使其在大肠杆菌得到表达,设计了带有SalⅠ,NotⅠ酶切位点的引物,以已构建好的质粒pGEM-T Easy/BRD7为模板,用PCR扩增出BRD7基因的完整阅读框架,并用SalⅠ,NotⅠ酶切PCR产物和原核表达载体PGEX-4T-2,然后用T4 DNA连接酶将其连接,得到重组表达质粒PGEX-4T-2/BRD7,经双酶切鉴定和测序验证,表达载体构建正确.重组表达质粒转化感受态大肠杆菌Jm105后用IPTG诱导,成功表达了一分子质量约为90 ku的融合蛋白;37℃诱导4 h后,SDS-聚丙烯酰胺凝胶(PAGE)电泳后,经扫描分析该融合蛋白产量占菌体蛋白总量28.48%, 蛋白质印迹(Western-blot)证实了该融合蛋白的表达获得成功.这为BRD7基因的蛋白纯化及抗体制备,进一步开展其功能研究奠定了基础.     

The expression of proUK in Escherichia coli: the vgb promoter replaces IPTG and coexpression of argU compensates for rare codons in a hypoxic induction model

The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.     

含蛋白转导域的SARA/SBD融合蛋白的原核表达、纯化及生物学活性鉴定

目的:SARA/SBD是纤维化形成过程中的负性调节因子。原核表达、纯化含反式激活蛋白(TAT)蛋白转导域(PTD)的TAT PTD-SARA/SBD融合蛋白,并鉴定其生物学活性。方法:将TAT PTD-SARA/SBD基因克隆入带His标签的原核表达载体pET-44a(+)中,转化大肠杆菌BL21,IPTG诱导表达,表达产物经Ni2+-NTA亲和层析柱纯化后,SDS-PAGE和Western印迹鉴定目的蛋白;用人腹膜间皮细胞系(HPMC),通过免疫细胞化学方法检测其穿膜能力,及与TGF-β1信号通路中Smad2因子的共定位情况。结果:用基因工程方法表达和纯化了TAT PTD-SARA/SBD融合蛋白,目的蛋白约占菌体总蛋白的20%左右,且以可溶形式表达,经Ni2+-NTA纯化后,所获蛋白纯度高于95%(HPLC归一法);功能学实验结果显示该蛋白能穿过胞膜,主要定位于胞核,且与Smad2因子具有核内共定位。结论:表达了TAT PTD-SARA/SBD融合蛋白,该蛋白具有生物学活性。     

大熊猫FOXL2基因的克隆、序列分析及表达

从大熊猫基因组中克隆了FOXL2基因,并对其进行序列分析及原核表达和真核表达.将FOXL2编码区序列克隆到原核表达载体pET-32a(+)中,转化大肠杆菌BL21,经IPTG诱导表达出FOXL2重组蛋白.成功构建了真核表达载体FOXL2-pcDNA3.1/V5-His C,并通过脂质体介导转染HEK293细胞,Western blot检测FOXL2蛋白表达.SDS-PAGE分析表明,FOXL2重组蛋白在诱导4h后表达量达到峰值,其大小约为58.9 kDa,Western blot分析结果显示重组蛋白能够被抗His单克隆抗体特异性识别.FOXL2基因的克隆及其表达为进一步进行FOXL2的活性检测以及应用研究奠定了基础.     

沙门菌外膜蛋白D的原核表达及多克隆抗体制备

目的:在大肠杆菌中表达沙门菌外膜蛋白(OMP)D,纯化后制备兔抗OMPD抗体。方法:用PCR方法从鼠伤寒沙门菌中扩增出ompD基因,并插入融合表达载体pET-28a(+)的多克隆位点,构建重组表达质粒pET28a(+)-ompD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,经IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行亲和层析纯化;以表达的OMPD蛋白免疫家兔,制备抗OMPD的多克隆抗体并进行鉴定。结果:扩增了ompD基因,测序证实正确后亚克隆于表达载体pET-28a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,经诱导在大肠杆菌中表达出相对分子质量为40×103的目的蛋白并进行纯化;纯化的OMPD免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶10000以上,且具有良好的特异性。结论:构建ompD基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗OMPD抗体,效价及特异性均良好,为进一步制备肠黏膜高亲和力疫苗奠定了基础。     

芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.