Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

INVOLVEMENT OF p38 MITOGEN-ACTIVATED PROTEIN KINASE IN E.coli-INDUCED U937 APOPTOSIS

Objective To investigate whether the effect of E. coli on U937 cell fines apoptosis is mediated via p38 mitogen-activated protein kinase （MAPK） activation. Methods The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry, p38 activities were detected by Western blotting. Results E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. Conclusion The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.

Dps Is a Stationary Phase-Specific Protein of <i>Escherichia coli</i>Nucleoid

Bacterial genomic DNA is highly organized into one or few compacted bodies known as nucleoid, which is composed of DNA, RNA and several DNA-binding proteins. These DNA-binding proteins require essential alterations in their expression during stationary phase of growth in order to re-spond to stressful environmental conditions. Dps (DNA-binding protein from starved cells) is one of such DNA-binding proteins, which accumulates most when E. coli cells reach to the stationary phase. Here, we have characterized Dps protein under various growth phases. Immunofluorescent microscopic observation reveals that Dps plays a key role in final round of genome compaction during the stationary phase. Similar results are also obtained by Western immunoblot analysis, after quantification of Dps protein from the exponential phase and early stationary phase nucleoid bound fractions, separated by sucrose density gradient centrifugation. Our results support the conclusion that Dps occupies more than half of the stationary phase nucleoid in E. coli.

A Simple Procedure for Extraction of Surface Protein of Salmonella Serotypes and Escherichia coli Strains Isolated from Poultry and Pigs

Salmonella and E.coli possess different surface protein structures that can induce protective immune responses.Identification of these proteins capacitates development of diverse applications in prevention and diagnosis that contribute to effectively control disease-causing enterobacteria pathogens such as Salmonella and E.coli.A simple procedure for obtaining protein complexes of Salmonella serotypes and E.coli is performed in this study.A sonication process with heat treatment of whole bacteria induced the release of protein complexes.Concentration of the protein extract was quantified using protein quantification Kits-Rapid,and protein complex profile was obtained by SDS-PAGE(Sodium dodecyl sulfate polyacrylamide gel electrophoresis)and silver staining.The concentrations of protein ranged from 29.45 to 45.35μg/mL in the Salmonella protein extracts,and from 25.35 to 36.72μg/mL in the E.coli protein extracts.Six major groups of proteins from E.coli(YfiO,NipB,OmpF,YfgL,Talc,YaeT)and four major groups of proteins from Salmonella(Flagellin,OmpA,Porin,SEF21)were preliminarily determined by a simple procedure of extraction based on the molecular weight.

The Wheat Pathogenesis Related Protein (TdPR1.2) Ensures Contrasting Behaviors to <i>E. coli</i>Transformant Cells under Stress Conditions

The pathogenesis-related proteins 1 (PR-1) gene family play important roles in the plant metabolism in response to biotic and abiotic stresses. The wheat TdPR1.2 has been previously isolated and characterized. Here we showed by bio-informatic analysis that TdPR1.2 contains six cysteine residues that are conserved between all PR-1 proteins tested. Using ScanProsite tool, we found that TdPR1.2 structure has a CRISP family signature 1 and 2 located at the C-terminal part of the protein. Those two domains are conserved in many identified PR1.2 proteins in plants. Moreover, SignalIP-5.0 analysis revealed that TdPR1.2 contains a putative signal peptide formed by 25 amino acids at the N-terminal extremity. The presence of this signal peptide suggested that the mature proteins will be secreted after the cleavage of the signal sequence. Further, we investigate the role of the TdPR1.2 proteins in the growth of Escherichia coli transformants cells under different abiotic stresses. Our results showed that the full-length form of TdPR1.2 enhanced tolerance of E. coli against salt and osmotic stress but not to KCl. Moreover, TdPR1.2 protein confers bacterial tolerance to heavy metals in solid and liquid mediums. Based on these results, we suggest that the TdPR1.2 protein could play an important role in response to abiotic stress conditions.

TAT-hEGF融合蛋白在E.coli BL21(DE3)中高效自我表达

为了获得TAT-hEGF融合蛋白在E.coliBL21(DE3)中高效表达,构建了原核表达载体pRSET-tat-hegf,将其转化E.coliBL21(DE3)得到重组工程菌BL21(DE3)/pRSET-tat-hegf。工程菌在无IPTG的诱导下实现了高效表达,TAT-hEGF融合蛋白的表达量占总菌体蛋白的45.6%,主要以包涵体形式存在。

在大肠杆菌中高效表达重组Protein A

本文构建了高效表达质粒pPA-3,其在大肠杆菌中表达的重组Protein A仅含天然Protein A的免疫球蛋白Fc段结合区,表达量达菌体可溶蛋白的20%。SDS-PAGE及Westernblot结果显示,重组Protein A的分子量有4种,即33、32.2、29.5和28.6kDa,其中33kDa与理论计算结果一致,推测其它分子量可能是由于胞内蛋白酶降解所致。一步亲和层析即可将重组Protein A从细胞裂解上清液中纯化出来。火箭电泳及酶联免疫分析结果表明,等蛋白量的该重组Protein A比天然Protein A能结合更多的免疫球蛋白。

Prokaryotic Expression of Antimicrobial Peptide CATH PR1–2 from the Skin of Paa robertingeri in Escherichia coli

The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin （CATH） PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low （3195.88 and 2838.34 Da, respectively）. Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid pET-32a. This reconstructed plasmid was then transfected into the expression vector E. coli BL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37℃ for 4 h. The antimicrobial activity assay using Staphylococcus aureus （Song） and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.

Development of an Indirect ELISA for Detection of T.solium Based on Recombinant 18 kDa Protein

The gene encoding the 18 kDa protein of Taenia solium metacestodes was amplified by RT-PCR and cloned into the pGEM-T vector for sequencing.The recombinant plasmid named pGEX-CE18 was constructed and transformed into E.coli BL21 for in vitro expression.SDS-PAGE and Western blot were employed for analyzing the recombinant protein,which was then used for development of an indirect ELISA for detection of anti-cysticercosis antibodies.The results showed that the recombinant protein of interest was 35 kDa in size,accounting for 28%of total bacteria proteins,and reacted with positive sera against cysticercosis.Using the newly-constructed indirect ELISA and a commercially available ELISA kit,paired analyses of 178 serum samples indicated that the concordant rate was 98.83%and the ELISA exhibited good specificity and sensitivity,supporting its utility and application for diagnosis of cysticercosis.

重组人血管内皮抑制素(rh-Endostatin)大肠杆菌表达体系发酵条件的优化

优化了重组人血管内皮抑制素的E.coli表达体系的发酵条件。利用E.coli表达体系得到了较高的产量,在9h左右的发酵周期内达到OD600值140,包涵体蛋白产量为3g/L。主要优化了异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)的终浓度、诱导时间、培养温度、补料控制方法等条件,并且在诱导后提高培养温度到40℃,在非常短的培养周期内达到了高密度培养的目的。利用E.coli表达,继而通过复性获得有活性的重组人血管内皮抑制素,成本低、生产过程稳定可控、得到的蛋白性质稳定,符合工业生产的需要。

乙脑病毒E蛋白中和表位与结核杆菌hsp70在大肠杆菌中的融合表达

根据大肠杆菌偏嗜性密码子原则,人工合成了乙脑病毒E蛋白C末端的中和表位,位于E蛋白上373-399 aa,通过EcoRⅠ和HindⅢ连接构建原核表达载体pET-32a-epitope。将hsp70通过HindⅢ和XhoⅠ连接到载体pET-32a-epitope上中和表位的3′端,构建融合表达载体pet-32a-epitope-hsp70。诱导表达后,将上清和沉淀分别进行SDS-PAGE,结果表明该融合蛋白以包含体形式存在。将包含体在8 mol/L的尿素中过夜溶解,然后将上清过柱,获得良好的纯化结果。融合蛋白的分子量为94 kD。Western blot显示该蛋白兼具对JEV和结核杆菌热休克蛋白70（M.Thsp70）的特异抗原性。

HBV PreS2-MBP融合蛋白在大肠杆菌中表达条件的优化

目的优化HBV PreS2-MBP融合蛋白在大肠杆菌中的表达条件。方法通过改变重组质粒的宿主菌、培养基、诱导温度、诱导剂、诱导剂浓度、诱导时间等条件,利用SDS-PAGE和BandScan凝胶分析软件,分析以上条件改变对表达产物表达量的影响。将重组质粒连续传100代,检测融合蛋白的表达稳定性。结果重组质粒在大肠杆菌BL21菌株、GS培养基、28℃、0.5mmol/L IPTG或1.5mmol/L乳糖诱导4h时,目的蛋白的表达量最高,占菌体总蛋白的43.5%。重组质粒传代100代,融合蛋白的表达稳定。结论已确定出了融合蛋白在大肠杆菌中表达的最佳条件。

扁蓿豆MrLEA2基因的克隆和原核表达

从扁蓿豆(Medicago ruthenica L.)幼苗中克隆到一个编码晚期胚胎发生丰富蛋白的基因MrLEA2,Pfam数据库检索表明其编码产物属于LEA_2蛋白家族。半定量RT-PCR分析发现MrLEA2在幼苗中表达水平不受非生物胁迫(脱水、高盐和低温)和脱落酸诱导。利用MrLEA2基因构建原核表达载体,在大肠杆菌中实现了过量表达。通过斑点试验和菌落计数实验,对过量表达MrLEA2蛋白的大肠杆菌细胞在高盐(0.5mol/L NaCl和0.5mol/L KCl)、55℃高温和-20℃冷冻胁迫处理下的生长存活情况检测发现,MrLEA2蛋白过量表达能够明显提高大肠杆菌对上述胁迫的耐受性。研究表明,MrLEA2蛋白对高盐和温度胁迫引起的细胞损伤具有保护作用。

烟夜蛾(Helicoverpa assulta Guenée)滞育激素基因在大肠杆菌中的表达

采用RT PCR技术从烟夜蛾(HelicoverpaassultaGuen啨e)滞育蛹中扩增到滞育激素(diapausehormone,DH)基因cDNA,将其与pET 30a(+)质粒连接后转化至大肠杆菌BL21中,测序结果表明,重组质粒插入片段编码序列完整,读框正确;重组工程菌用IPTG诱导表达后,SDS聚丙烯酰胺凝胶电泳检测到一条约21kDa大小的外源蛋白,它与DH的大小相应.DH基因原核表达载体的成功构建为大量获取DH重组蛋白、研究DH的结构及功能奠定了基础.

乙型脑炎病毒E蛋白N端片段在大肠杆菌中的表达

利用 PCR扩增乙型脑炎病毒 E蛋白基因 5′端片段 ,克隆入原核表达载体 p ET-2 8a,转化大肠杆菌 BL2 1 ( ED3 )。经 IPTG诱导表达后 ,SDS-PAGE分析表达产物。结果表明 ,所表达的E蛋白片段的分子量约为 3 .4万 ,表达量约占菌体总蛋白的 3 5%。 JEV E蛋白片段在大肠杆菌中的成功表达 ,为制备 JE实验室诊断抗原和分析

脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选

应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。

金龟子绿僵菌(Metarhizium anisopliae HN1)几丁质酶与谷胱甘肽S-转移酶GST在大肠杆菌中的高效融合表达

使用RT-PCR方法,从高毒力金龟子绿僵菌Metarhizium anisopliaeHN1中,克隆得到一个全长为1 275 bp的几丁质酶基因,经Blast分析此基因序列与M.anisopliaeE6的chi1基因(AF02749)同源率为96%。将此基因克隆到pGEX-6p-1载体上,使之与载体上一个约26kD大小的谷胱甘肽S-转移酶(GST)相连,构建pGEX-chi融合表达载体,转化到大肠杆菌(Escherichia coli)BL 21中,经SDS-PAGE结果分析显示:表达出的融合蛋白大小为68 kD,此目的蛋白占表达总量的64.5%。经破碎处理后可检测到几丁质酶活性。