Prokaryotic expression of Staphylococcus aureus Panton-Valentine leukocidin and its cytolytic activities to human polymorphonuclear neutrophils

AIM:Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus epidemiologically associated with the often-lethal necrotizing pneumonia. Until now, the mechanisms of pathogenesis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult. In the present study, we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro, which provides an experimental basis for the further studies of its biological function and its toxicity in pneumonia. METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by high-fidelity PCR was cloned into prokaryotic expression vector pET22b(+), and the vector was transformed into BL21 (DE3)plysS to construct a prokaryotic expression system. The integrity of the opening-reading frame of each construct was verified by DNA sequencing. The recombinant PVL (rPVL) was induced by1.0 mmol/L IPTG. The expressed products were identified by SDS-PAGE and the fusion proteins (6His-LukS-PV and 6His-LukF-PV) were purified from lysates of transfected E. coli cells by affinity chromatography on nitrilotriacetic acid columns. The cytolytic activity was tested by incubation of rPVL with human polymorphonuclear neutrophils (PMNs) in vitro. RESULTS:The nucleotide sequence of the cloned PVL gene was the same as that of reported in GenBank. E. coli BL21 (DE3)plysS containing recombinant vectors grow at 37℃ causes some proteins to accumulate as inclusion bodies, while incubation at 30 ℃ led to a significant amount of soluble active proteins which accounted for about 31.7% of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD, and the LukF-PV protein was about 35 kD. The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated. CONCLUSION:The genes of lukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+) and expressed in E. coli respectively, which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.     

Prokaryotic expression and functional study of human high mobil ity group box 1 protein

AIM:To construct the prokaryotic expressio n plasmid of His-tagged human high mobility group box 1 fusion protein (hHMGB1) and to express the fusion protein in E.coli for the affinity purification.METHODS:The cDNA coding region of HMGB1 was amplified by PCR fr om pGEX4T-HMGB1 and cloned into a modified pET14b vector following the routine p rocedure.After identification by enzyme digestion,PCR and sequencing,the plas mid was transformed into BL21 (DE3) competent cells,and the His-HMGB1 fusion pro tein was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPT G),and further purified by Ni-NTA affinity chromatography.The protein was f il tered for sterilization and used to stimulate human umbilical vein endothelial c ells (HUVECs).24 hours later,the cultured supernatant of HUVECs was collected for the detection of cytokines/chemokines with LiquiChip system.RESULTS:The His-HMGB1 fusion protein expression plasmid was ide ntified by enzyme digestion and sequencing.The purified His-tagged fusion prote in was analyzed by SDS-PAGE and Western blotting with specific anti-His antibody .It was found that the production of IL-8 from HUVECs was highly induced in a d ose-dependent manner by HMGB1.CONCLUSION:The His-tagged HMGB1 fusion protein expression plasm id was successfully constructed,and purified.Recombinant HMGB1 protein has a h igh bioactivity on the induction of cytokines in HUVECs,which may significantly facilitate the future study of HMGB1 biological functions.     

Prokaryotic expression and preparation of GST/BFRF3 fusion protein of Epstein-Barr virus for screening nasopharyngeal carcinomas

AIM: To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsid antigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS: DNA extracted from the B95-8 cells was used as the templates. Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene, and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector. The recombinant plasmid was transformed into E.coli BL21 (DE3). The expression of GST/BFRF3 fusion protein was induced by IPTG, identified by both SDS-PAGE and Western blotting, and then purified by glutathione-sepharose beads. The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS: The GST/BFRF3 fusion protein was successfully expressed in E. coli. The molecular weight of the product was approximately 44 kD. The recombinant fusion protein GST/BFRF3 showed good immunoreactivity. A novel ELISA was established using GST/BFRF3 protein. Serum samples collected from the NPC patients and healthy controls were tested by this ELISA. The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％, respectively.CONCLUSION: The recombinant protein GST/BFRF3 is expressed in E.coli, and it has diagnostic value for screening of NPC patients.     

瓜类褪绿黄化病毒p22 基因在大肠杆菌中的表达及抗血清的制备

 以被瓜类褪绿黄化病毒（Cucurbit chlorotic yellows virus,CCYV）侵染的甜瓜叶片为供试材 料,采用RT-PCR 方法克隆其P22 蛋白基因,并将其连接到原核表达载体pGex-4T-3 上,PCR 验证及克隆 测序确定开放阅读框的正确性。将重组载体pGexp22 转化大肠杆菌BL21 菌株,诱导表达,SDS-PAGE 分 析表明,经IPTG 诱导,p22 基因在大肠杆菌BL21 中得到了高效表达。以表达的蛋白作为抗原,免疫家 兔,制备了CCYV P22 的特异性抗血清。ACP-ELISA 检测结果表明,血清效价高达1.28 × 10⁵。Western blot 检测甜瓜叶片,结果表明抗血清能够特异性地检测CCYV 侵染的甜瓜叶片中的CCYV P22 蛋白。     

‘三棱榄''橄榄果实香气成分分析

1 材料与方法选取广东优良鲜食橄榄品种‘三棱榄’,2001年12月6日采样,采用固相微萃取法(SPME)富集香气成分(鲜橄榄果肉于15℃下捣碎后取样1.0 g放入4 mL聚四氟乙烯硅橡胶垫密封螺口玻璃瓶中,插入100μm聚二甲基硅氧烷纤维头于室温25-30℃顶空取样2 h),用美国Finnigan TRACE GC-MS气相色谱-质谱联用仪进行分析。气相色谱柱为DB-1弹性毛细管柱30 m×0.25 mm,载气为He(99.99％),流速1.0mL/min。程序升温从40℃开始先保持10 min,后以2℃/min的升温速率升至150℃保持10 min。质谱条件:电子能量70 eV,离子源温度250℃,质量范围35-450 aum,不分流进样。2002年12月18日采样重复分析。     

Expression and purification of re-constructed human CRP and observation of its internalization into HeLa cells

AIM: To construct prokaryotic expression vector for human C-reactive protein (CRP), to acquire the functional fusion protein purified from BL21(DE3) transformed with vector pET14b/EGFP-hCRP, and to observe the internalization of the fusion protein his-EGFP-CRP into tumor cell line HeLa. METHODS: CRP gene sequence was amplified with the vector p91023/CRP as template by PCR, and was inserted into vector pET14b/MCS-EGFP-(N)36 to construct prokaryotic expression vector. The E. coli cells BL21(DE3) transformed with the re-constructed vector pET14b/EGFP-hCRP was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the expressed protein his-EGFP-CRP were purified with affinity chromatography method and refolded with gradient filtration. The HeLa cells were observed under the fluorescence microscopy after the addition of purified renature protein. RESULTS: The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated the construction of vector pET14b/EGFP-hCRP was correct; the SDS-PAGE showed that the transformed E. coli cells could be induced to express the fusion protein his-EGFP-CRP and the purification of proteins were successful. We could found fluorescent signal around the cell membranes, in the cytoplasm and nuclei in the observation of the HeLa cells incubated with his-EGFP-CRP. CONCLUSION: The prokaryotic expression vector for human CRP linked with his and EGFP coding sequence is successfully constructed. The fusion protein his-EGFP-CRP is purified and refolded. The reconstructed protein expressed by prokaryotic cells adheres to the membrane of tumor cell HeLa and is internalized into the cytoplasm and nuclei of the cells.     

Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 μg/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.     

柑橘溃疡病致病基因PthA核定位信号肽抗血清制备及其抑菌的研究

 用PCR法扩增位于柑橘溃疡病菌致病基因pthA C-末端的3个核定位信号序列,并将其克隆到原核表达载体PET32a(+)上,经双酶切及核酸序列测定重组质粒(PthA-NLS),其序列与GenBank中pthA的相关序列有99.9%的同一性。重组质粒转化大肠杆菌BL21(DE3)后诱导了重组多肽的表达,并用Ni2+-NTA纯化柱得到了48kD的纯化重组多肽。把重组多肽注入免疫Balb/c小白鼠,制备了相应的抗血清,Western Blotting和ELISA分析结果表明,抗血清可特异地结合重组多肽,亦可识别溃疡病菌PthA天然蛋白,获得的抗血清可以用于柑橘溃疡病的检测。利用抗血清与溃疡病菌混合接种离体冰糖橙叶片,发现抗血清能推迟溃疡病菌的致病过程,且病斑比对照小,但未能达到抗病的程度。pthA基因末端核定位信号序列的克隆、原核表达及抗血清的制备为进一步研究pthA的致病机理和研发溃疡病快速分子检测技术奠定了基础。     

欢迎订阅2006年下列期刊

AIM: To construct prokaryotic expression vector of His-tagged human IP-10 for further study of its biological function in the inflammatory response. METHODS: The coding sequence of IP-10 lacking signal peptide was amplified from human lung cDNA library by polymerase chain reaction (PCR) and the fragment was cloned into pET-14b plasmid for the construction of His-tagged fusion protein expressing vector, pET-14b/IP-10. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli, BL21 (DE3). The expression of His-tagged fusion protein was induced with IPTG and purified with Ni+-NTA affinity chromatography. Then the chemotactic activity of IP-10 was determined by transwell migration assay on THP-1 cells. RESULTS: The construction of pET-14b/IP-10 recombinant vector was proved by enzyme digestion and sequencing. The fusion protein IP-10, which was purified by a routine Ni+ affinity method, had an activity on the induction of cell migration of THP-1. CONCLUSION: We successfully construct IP-10 fusion protein expressing vector and get the fusion protein with high bioactivity, which provides essential materials for the future studies on IP-10. V     

Construction of human neuronal nitric oxide synthase expression system in Escherichia coli

AIM: To construct a high-level expression system of recombinant human neuronal nitric oxide synthase (hnNOS) full-length enzyme in Escherichia coli. METHODS: The coding sequence of hnNOS full-length was firstly amplified by PCR, and then ligated into the expression vector pCWori+. The recombinant plasmid was transformed into Escherichia coli BL21 for high-level expression. After having been checked with Western blot, the enzyme was used for large-scale culture and purification. Finally, the property of the enzyme was determined by spectrophotometric method. RESULTS: The constructed expression system could give a yielding of 3 mg/L initial culture. CONCLUSION: The expression system constructed is fully sufficient to express the active human neuronal nitric oxide synthase.     

Construction of prokaryotic expression vector of human angiogenesis inhibitor arresten and its expression in E.coli

AIM:To construct prokaryotic expression vector of human angiogenesis inhibitor arresten gene and express recombinant arresten in Escherichia coli.METHODS:Human arresten gene was amplified from recombinant plasmid pGEM-Arr with polymerase chain reaction (PCR), and then cloned into prokaryotic expression vector pRSET by means of recombinant gene technology. The recombinant plasmid pRSET-Arr was transformed into E.coli BL21(DE3), and recombinant arresten was expressed in the bacteria under induction of IPTG. The expressed products were detected by SDS-PAGE analysis.RESULTS:Restriction analysis indicated that the arresten gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant arresten was successfully expressed in Escherichia coli; its molecular weight was about 26 kD and its amount was approximately 30% of total bacterial proteins.CONCLUSION:The successful construction of prokaryotic expression vector containing human arresten gene and the effective expression of recombinant arresten in Escherichia coli laid the foundation for further study on its biological functions.     

High and stable expression of an analog of human basic fibroblast growth factor in Escherichia coli

AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET-3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3) plysS. After induction by IPTG, the analog was obtained and analyzed by SDS-PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein's bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E.coli.     

香蕉果实转录因子MaWRKY1基因的原核表达和多克隆抗体制备

MaWRKY1是从香蕉果实中克隆出的一个转录因子基因。为进一步研究该基因的功能,制备了MaWRKY1多克隆抗体。选取MaWRKY1基因全长中N端第168 ~ 400个氨基酸之间的包括两个WRKYGQK保守域的cDNA序列,构建了原核表达载体pET-MaWRKY1,并转化到大肠杆菌BL21中诱导表达菌体蛋白。SDS-PAGE电泳检测结果表明,His-MaWRKY1融合蛋白成功获得了高效表达,分子量在26 kD左右。His-MaWRKY1融合蛋白经过Ni-NTA琼脂糖凝胶树脂纯化,SDS-PAGE制备胶割胶富集,电洗脱法纯化后得到的纯化蛋白浓度达到0.5 mg • mL-1。经对新西兰兔进行5次免疫,获得了多克隆抗血清,采用免疫吸附方法对抗血清进行了纯化。将纯化后的抗体通过间接酶联免疫（ELISA）和蛋白质印迹（Western blot）分析,表明所制备的抗体具有很好的效价,效价比为1︰160 000,同时具有良好的灵敏度和特异性。进一步提取香蕉不同组织总蛋白,Western blot检测显示,在分子量26 kD左右处出现特异的蛋白质条带,证明所制备的抗体可以与香蕉WRKY蛋白特异性结合,并且低温可以诱导香蕉果实中MaWRKY1蛋白表达,暗示MaWRKY1蛋白表达可能与果实耐冷性有一定的关系。     

温州蜜柑萎缩病毒小外壳蛋白基因克隆与原核表达及其抗体制备

 用RT-PCR方法,从采自重庆奉节的‘宫本’柑橘的2个样品中扩增出温州蜜柑萎缩病毒（Satsuma dwarf virus,SDV）SDV RNA2的3′末端,长度为975 bp,与报道的SDV S-58 3′末端序列同源性分别为98.7%和98.4%。根据获得的序列设计引物,以含有SDV FJ 3′末端序列的质粒为模板,PCR扩增获得大小为 654 bp的SDV-FJ小外壳蛋白（Small coat protein,CPS）基因产物。构建了CPS与GST融合表达载体PGEX-CPS,在37 ℃、1.0 mmol · L-1 IPTG条件下诱导表达,SDS-PAGE电泳分析表明成功表达出分子量约为42 kD的GST-CP融合蛋白。以表达的融合蛋白为抗原免疫家兔,制备的抗血清的效价为1/12 800。用获得的抗体进行组织印迹分析表明,SDV在柑橘叶柄基部韧皮部维管束区域含量最高。     

百合无症病毒16kD基因的克隆、原核表达及抗血清制备

以感染百合无症病毒(LSV)的百合叶片为试材,克隆LSV16 k D基因,连接到原核表达载体p ET-28a(+)上。将获得的重组质粒p ET-28a(+)+16 k D转化大肠杆菌BL21(DE3),经IPTG诱导得到了高效表达的16 k D蛋白,融合蛋白分子量约为20 k D。融合蛋白经过镍柱纯化后作为抗原免疫注射小鼠,制备得到16 k D蛋白抗血清。Western blot分析显示所制备的抗血清与诱导表达的融合蛋白发生特异性反应;通过ELISA检测和RT-PCR检测百合样品,证实制备的抗血清与LSV侵染的百合叶片发生了相同的特异性反应。结果表明,目的蛋白表达成功,所制备的抗血清具有特异性,可用于LSV的快速检测、免疫组织化学以及16 k D蛋白功能研究。     

双孢蘑菇多酚氧化酶的分离、纯化及特性分析

 通过分步盐析,DEAE-Cellulose-52阴离子交换层析和Sephadex G-100凝胶柱层析,对双孢蘑菇中的多酚氧化酶（PPO）进行了分离纯化,并对在分离纯化过程中蛋白得率及酶活性进行了测定。最终得到纯化倍数为103.2,比活为566.57 U · mg^-1,蛋白得率为0.47%的酶液。经SDS-PAGE电泳检验,所得到的PPO呈单一蛋白带,分子质量为25.5 kD。利用扫描探针显微镜对该酶的分子形态进行了观察,发现双孢蘑菇中的PPO分子呈椭球状,分子高度在8 ~ 12 nm之间。     

Effect of hemolysin protein HlyX of Leptospira interrogans serovar Lai on permeability of vascular endothelial cells

AIM: To clone and express the hemolysin gene hlyX of Leptospira interrogans serovar Lai and to investigate the effect of the expression product on the permeability of human umbilical vein endothelial cells (HUVECs).METHODS: The recombinant plasmid pET-hlyX was constructed by inserting the hlyX gene into prokaryotic expression vector pET32a(+), and transformed into E.coli BL21(DH3) to express the fusion protein Trx-HlyX with a His-tag.The fusion protein was purified using HisTrap affinity columns.The permeability of the monolayer HUVECs was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin.Flow cytometry and Hoechst 33258 staining were applied to measure the apoptotic rate of HUVECs after incubation with Trx-HlyX.RESULTS: The recombinant plasmid pET-HlyX was successfully constructed and the fusion protein Trx-HlyX was highly expressed.Compared with the control cells, the purified recombinant protein Trx-HlyX significantly increased the permeability of transfected cells and promoted apoptosis of HUVECs (P<0.05).CONCLUSION: The recombinant plasmid pET-hlyX highly expresses the fusion protein Trx-HlyX.Purified protein Trx-HlyX influences the permeability and has cytotoxicity on HUVECs.     

Expression of recombinant human tumor suppressor NDPK-A in E. coli

AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6×His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6×His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni⁺-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49.6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6×His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.     

黄瓜动蛋白特异肽段的原核表达和多克隆抗体制备

 以黄瓜果实总RNA为模板,通过RT-PCR获得了动蛋白基因CsKF1和CsKF2的cDNA序列,进行测序,构建T-easy载体。SMART在线预测CsKF1和CsKF2均含有动蛋白（Kinesin）约340个氨基酸残基的马达区保守序列（moter domain）。体外表达蛋白的马达区和非马达区,将带有His标签的CsKF1-N、CsKF2-C、CsKF1-M、CsKF2-M融合蛋白通过E. coli BL21（DE3）表达,摸索不同诱导条件下目的蛋白的表达和纯化。其中His-CsKF1-N、His-CsKF2-C、His-CsKF1-M原核表达蛋白的分子量分别为25、30和40 kD,均以0.1 mmol · L^-1 IPTG,22 ℃ 6 h诱导表达效果最好。His-CsKF2-M原核表达蛋白的分子量约38 kD,以18 ℃ 8 h诱导表达效果最好。融合蛋白His-CsKF1-N和His-CsKF2-C经过亲和纯化后作为抗原,通过5轮免疫注射兔子后,取心脏血作为抗血清,通过AminoLink Plus kit试剂盒亲和纯化得到多克隆抗体anti-CsKF1-N和anti-CsKF2-C。经过Western blot分析表明多克隆抗体具有特异性,可与抗原特异结合。