Direct and rapid electrochemical biosensing of the human interleukin-2 DNA in unpurified polymerase chain reaction (PCR)-amplified real samples

Electrochemical detection of polymerase chain reaction (PCR)-amplified human interleukin-2 (IL-2) coding DNA sample (399bp size) without any purification and pre-treatment is described. To achieve this goal, a sensor was made by immobilization of a 20-mer oligonucleotide (chIL-2) as the probe on the pencil graphite electrode (PGE). This probe is related to the antisense strand of human interleukin-2 gene. The results showed that the electrode could effectively sense the PCR product of human interleukin-2 DNA by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. In order to inhibit PCR components interfering effects and improve biosensing performance, various factors were investigated. We found that the desorption of non-specifically adsorbed components of the unpurified PCR samples from PGE surface is easily achieved by washing of the electrode in washing solution for about 300s. The effectiveness of this procedure was confirmed using purified PCR samples. The selectivity of the sensor was assessed with negative control PCR sample and seven different non-complementary PCR products corresponding to 16S rDNA (bigger than 1500bp) of various bacterial genuses. Diagnostic performance of the biosensor is described and the detection limit is found to be 69pM. The reliability of the electrochemical biosensing results was verified by electrophoresis of the PCR products.     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

拟南芥血红蛋白1(AtGLB1)与过氧化氢的相互作用

拟南芥的血红蛋白1(AtGLB1)属于非共生的血红蛋白。在低氧胁迫中对植物细胞中过氧化氢(H2O2)内稳态的维持起了很重要的作用。为了检测AtGLB1与H2O2能否直接相互作用,我们扩增了拟南芥的AtGLB1基因,并将其克隆到原核表达质粒pET32a中,测序鉴定正确后转化大肠杆菌BL21。IPTG诱导目的蛋白表达后,镍离子亲和层析柱(Ni2+-NTA)纯化了靶蛋白。体外表达的氧合的AtGLB1能与H2O2直接相互作用。因此,与H2O2反应可能是AtGLB1清除低氧胁迫下产生的H2O2的一种方式。     

嗜吞噬细胞无形体aph0653的克隆表达及重组蛋白的抗原性分析

目的:克隆表达嗜吞噬细胞无形体(Anaplasma phagocytophilum,AP)APH0653基因,并对表达产物进行抗原性分析。方法:以嗜吞噬细胞无形体基因组DNA为模板,使用特异性引物,PCR扩增APH0653基因并克隆入原核表达载体进行表达。使用镍柱亲和层析纯化重组蛋白,并应用免疫印迹方法检测APH0653与嗜吞噬细胞无形体感染血清的免疫反应性。结果:菌落PCR、DNA测序和SDS-PAGE蛋白凝胶电泳表明APH0653基因已成功克隆入原核表达载体,并可诱导表达重组蛋白。免疫印迹实验表明AP阳性血清可识别重组蛋白APH0653,并产生明显的特异性条带。结论:嗜吞噬细胞无形体APH0653蛋白可在原核表达系统中高效表达,且重组蛋白具有较好的抗原性。     

重组人FOXL2基因的原核表达和纯化研究

目的通过pET32a（＋）原核表达载体,表达重组人叉头框蛋白L2（human forkhead box12,FOXL21）。并且进行纯化和鉴定。方法从正常人血液中提取基因组DNA,利用PCR扩增FOXL21目的基因片段,构建FOXL21原核表达重组质粒[pET32a（＋）-FOXL21]并转化E．coli的BL21（DE3）菌株,IPTG诱导重组蛋白表达,经HisTrap FF亲和层析柱纯化,再通过SDS—PAGE和Western印迹鉴定。结果成功克隆到大小为1131bp的人源FOXL21基因片段并准确插入表达载体pET32a（＋）,0．1mmol／LIPTG诱导转化菌8h可表达大量的FOXL21蛋白,并可经HisTrap FF柱亲和层析得到高度纯化。结论成功获得纯化的66kD重组人FOXL21蛋白,为后续进行FOXL21蛋白的功能研究奠定了基础。     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

人工锌指核酸酶的设计与表达纯化

设计表达了四个锌指核酸酶,用于切断人基因组中的rRNA基因家族的内部转录间隔序列,造成双链断裂,以此提高针对多位点基因打靶的效率,为后续基因打靶应用于基因治疗研究奠定基础。首先,在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点,根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA,分别克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFP,转化大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时,将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFN,转化到大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。     

Molecular cloning, expression, and purification of SARS-CoV nsp13

The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using TA cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M.bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

黄颡鱼自噬相关基因 LC3B和Beclin1的原核表达及多克隆抗体制备

为深入研究黄颡鱼Pelteobagrus fulvidraco自噬的发生过程和机制, 从黄颡鱼cDNA中克隆获得372和1344 bp的黄颡鱼LC3B(PfLC3B)和Beclin1(PfBeclin1)基因编码序列, 并成功构建了pET32a(+)-PfLC3B和pET32a(+)-PfBeclin1重组表达载体, 分别采用亲和层析和包涵体纯化的方法得到了纯度较高的重组PfLC3B和PfBeclin1蛋白; 以重组PfLC3B和PfBeclin1蛋白免疫Balb/C小鼠, 制备多克隆抗体。通过Western blot鉴定了PfLC3B和PfBeclin1抗血清可以分别特异性识别重组PfLC3B和PfBeclin1蛋白; PfLC3B抗血清可以特异性识别黄颡鱼内源性LC3-Ⅰ和LC3-Ⅱ蛋白, PfBeclin1抗血清可以特异性识别黄颡鱼内源性Beclin1蛋白。蛋白水平的组织分布显示, 黄颡鱼LC3和Beclin1蛋白在肝脏中表达水平均较高, 在肾脏和脾脏中的表达水平较低。综上所述, 研究成功制备了黄颡鱼LC3B和Beclin1的多克隆抗体, 为深入研究黄颡鱼自噬机制提供了有力工具。     

Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus

The DNA polymerase gene of Thermococcus marinus ( Tma ) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI- Tma , was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli . PI- Tma cleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangs with 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI- Tma was optimal when present in 50 mM glycine–NaOH (pH 10.5), 150 mM KCl and 12 mM MgCl₂ at 70 °C.     

Lys_(10)-PAI-1融合蛋白在大肠杆菌中的表达、纯化和凝胶阻滞实验

用PCR方法构建 10个赖氨酸与纤溶酶原激活剂抑制物 1的融合基因Lys10 PAI 1,并克隆于pET2 8a(+ )和pET32a(+ )原核表达载体 .将重组表达质粒pET32 PAI和pET2 8 PAI转化大肠杆菌BL2 1(DE3) .IPTG诱导后可获得分子量分别为 6 3kD和 4 3kD的目的蛋白 ,表达蛋白占菌体蛋白2 0 %以上 ,大多数重组蛋白以不溶形式存在 .表达产物经变性、复性、超滤、透析和亲和层析等步骤 ,可以得到纯化的Trx·PAI 1和rPAI 1重组蛋白 .Western印迹结果表明 ,目的蛋白具有人PAI 1的抗原性 .凝胶阻滞实验发现 ,纯化的重组蛋白在一定条件下 ,可以与质粒结合 ,使质粒的迁移率明显改变 .研究结果表明 ,Trx·PAI 1和rPAI 1有希望成为受体介导基因转移的配体     

金黄色葡萄球菌肠毒素B基因的克隆和在大肠杆菌中高效表达

利用PCR方法从金黄色葡萄球菌TSTw基因组DNA中扩增出约700bp的DNA片段,将之克隆到pGEM7Zf(+)载体上并转化大肠杆菌 DH5α菌株。重组质粒的测序结果表明克隆到了seb基因,它含有717bp（不包括N端81bp的信号肽编码区）,其核苷酸序列与文献报道完全一致。将其连接于表达载体7ZTS上,转化到大肠杆菌JM109（DE3）内。表达的SEB占总蛋白33.5%。      

立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

目的：克隆表达立氏立克次体（Rickettsia rickettsii）外膜蛋白H基因（ompH）片段并对其进行免疫原性分析。方法：采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果：获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论：pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

人IL-6受体胞外区真核表达载体的构建及体外表达

目的构建人IL-6受体（IL-6R）胞外区真核表达载体,检测其在体外培养细胞中的表达。方法利用PCR扩增IL-6R胞外区,克隆到pcDNA3．1（＋）中,用双酶切、测序鉴定。重组质粒通过脂质体转染HL-60细胞,用G418进行筛选,利用Western印迹检测IL-6R蛋白表达。结果PCR扩增出1218bp的目的片段,双酶切和测序结果显示重组质粒正确。Western印迹结果显示转染细胞能够表达目的蛋白。结论成功构建了人IL-6R胞外区真核表达载体,并且能够在真核细胞中表达。     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

美洲大蠊3-磷酸甘油醛脱氢酶基因的克隆及表达

旨在通过5’-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础.通过3’-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用Histag抗体Western blotting验证.结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸.序列分析显示,该蛋白与家蚕GAPDH相似性为89％,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白.