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.     

马铃薯卷叶病毒缺失突变CP基因的原核表达及抗血清的制备

 利用RT-PCR扩增马铃薯卷叶病毒（PLRV）外壳蛋白（CP）基因（PLRV-CP）,回收大小约630 bp的特异性扩增片段并进行T-A克隆,测序表明该基因长度为627 bp,与已报道的36个PLRV-CP基因的核苷酸序列的同源性大于96%。以pBAD/Thio-TOPO为起始载体,构建了PLRV-CP基因的原核表达载体pBAD-LRCP。以pBAD-LRCP为模板,用PCR法删除了该基因富含精氨酸稀有密码子的第52 ~ 177核苷酸,获得了PLRV缺失突变CP基因的原核表达载体pBAD-LRCP-126。用阿拉伯糖诱导工程菌TOP10（pBAD-LRCP-126）,获得了34 kD的诱导表达的融合蛋白（重组CP）。用镍离子亲和层析法从包涵体中纯化出了高纯度的重组CP,用纯化的重组CP作抗原免疫家兔获得了PLRV特异性的抗血清,间接ELISA检测显示效价为1︰12 800。本研究结果为利用重组CP作抗原大量制备PLRV抗血清奠定了基础。     

筇竹CBF1基因的原核表达和多克隆抗体的制备

以筇竹（Qiongzhuea tumidinoda Hsueh et Yi）叶片为试材,采用RT-PCR方法克隆其CBF1基因,并将CBF1连接到原核表达载体pET32a（+）上,经克隆测序确定所构建的重组载体pET32-QZ开放阅读框正确。将重组载体pET32-QZ转化大肠杆菌Rosetta2（DE3）菌株,经IPTG诱导表达,SDS-PAGE凝胶电泳,考马斯亮蓝染色,证明CBF1蛋白得到了高效表达,所表达蛋白是大小约为45 kD的融合蛋白。经镍柱纯化后作为抗原免疫家兔,制备CBF1蛋白特异性抗血清。所制备的多克隆抗体能够与融合蛋白和经冷诱导的筇竹叶片总蛋白在25 kD处出现杂交条带。上述结果表明,表达的目的蛋白可用于免疫组织化学、蛋白质印迹检测。     

Prokaryotic expression and identification of a small peptide of osteopontin

AIM: To express osteopontin 13 peptide (OPN 13) in E.coli, and to test the biological activity of the purified products. METHODS: cDNA fragments containing RGD sequences were cloned into prokaryotic expression vector pET-32c(+) including His coding sequence to construct pET-32c-OPN 13 plasmid. E.coli DH5α transformed by pET-32c-OPN 13 plasmid was induced by IPTG at different concentrations for different times to identify the optimal induction condition. Expressed His-OPN 13 fusion protein was purified via Ni-NTA His Bind Resin metal chelation chromatography, and detected by VSMCs adhesion and migration analysis. RESULTS: His-OPN 13 fusion protein was expressed in soluble manner. The fusion proteins were purified via Ni-NTA His Bind Resin affinity chromatography. His-OPN 13 fusion protein specifically inhibited adhesion and migration of VSMCs stimulated by osteopontin in dose-dependent manner. CONCLUSION: The OPN 13 peptide is successfully expressed in E.coli DH5α. The purified His-OPN 13 fusion protein could inhibit the adhesion and migration of VSMCs stimulated by osteopontin.     

Purification of an anti-HBsAg scFv and measurement of its affinity constant

AIM: To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6×His tag, and to determine the affinity constant of the purified recombinant product.METHODS: Solubilizing in buffers containing urea or guanidine hydrochloride(GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni²⁺ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA. RESULTS: The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached(61.08±1.45)%. The affinity constant of the polished scFv was confirmed to be(2.30±0.32) ×10⁷ L/mol. CONCLUSION: The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro. The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.     

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     

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。     

Cloning and expression of the metalloproteinase domain of human von Willebrand factor-cleaving protease

AIM: To clone and express the metalloproteinase domain of human von Willebrand factor-cleaving protease (vWF-cp). METHODS: The metalloproteinase domain of human vWF-cp, amplified from the plasmid containing the vWF-cp cDNA gene by using polymerase chain reaction, was cloned into pUC18, and its accuracy was verified by sequencing. Then the domain was inserted into the multiclone site of pET28a(+) and included a 6×His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified by using a Ni-NTA column and confirmed by Western blot. RESULTS: Comparison of the nucleotide sequence of our cloned domain with the GenBank sequence revealed no difference. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of total bacteria protein in inclusion body. Western blot demonstrated that it possessed high specificity. CONCLUSION: The metalloproteinase domain of vWF-cp was high efficiently expressed in Escherichia coli. This might contribute to the further study of the relationship between its structure and function.     

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.     

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。     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Gene cloning,prokaryotic expression,purification and characterization of CAD-caspase activated DNase

AIM：To construct a the plasmid pCool-GST-ICAD/CAD that expresses the CAD protein with biological function in E.coli BL21(DE3)strain.METHODS：CAD was amplified by PCR and inserted into pCool-GST-ICAD to create pCool-GST-ICAD/CAD.The plasmid was checked and transfected into BL21 for expression.The expressed protein were purified by using the GST affinity column,ion exchange and the gel filtration.Protein purity was checked by SDS-PAGE.CAD activity was checked by DNA fragmentation test.RESULTS：Plasmid pCool-GST-ICAD/CAD was constructed as design.The expression of GST-ICAD/CAD protein complex at mg level was observed.After purification,ICAD/CAD protein complex were obtained.Two clear protein bands were shown on SDS-PAGE.The DNA fragmentation test shows that the purified CAD protein indeed had the nonspecific DNase activity.CONCLUSION：Cloning and purification of CAD protein was successful with intact biological activity.     

Cloning and expression of Leptospira strain 017 hemolysin HlyX gene and its cytotoxicity

AIM: To clone HlyX gene and observe its expression and cytotoxicity.METHODS: The HlyX gene was amplified from Leptospira strain 017 genome by the polymerase chain reaction (PCR) and through enzyme digestion, and cloned into pET32a(+), then transformed into E.coliJM109. After induced with IPTG, the target protein was immunized to New zealand white rabbit. Western blotting was used to identify the immunogenicity of the expressed protein. The purified and renatured protein was acted on ECV304 cells to detect its cytotoxicity by examining the release of LDH and NO from the cells. RESULTS: The full length of the HlyX gene about 1 179 bp was obtained by PCR. The recombinant plasmid was identified by enzyme digestion, PCR and DNA sequencing. After induced with IPTG, the expressed protein existed in the form of inclusion bodies about 64 kD, which was consistent with the expected size of the fused protein. After immunity, the titre of the multiclonal antibody reached 1∶〖KG-*2〗64 000 by ELISA. Western blotting analysis found a positive band specifically in the target protein position. The release of the LDH and NO in the ECV304 cells treated with HlyX fusion protein showed significant increase compared with the control group (P<0.01). CONCLUSION: HlyX gene is expressed successfully in E.coli JM109, and the expressed products shows cytotoxicity.     

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.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Expression of recombinant quadrivalent Aβ1-15 and its immunogenicity identification

AIM:To prepare the second-generation Alzheimer disease vaccine of quadrivalent tandem Aβ1-15 and to explore its immunogenicity.METHODS: The 4×Aβ15 gene was amplified by PCR with the recombinant plasmid pcDNA-4×Aβ15 as a template, which was prepared by our research group before. The 4×Aβ15 gene was then cloned to the pGEX-4T-2 plasmid. The fused GST-4×Aβ15 protein was expressed when the bacteria containing the recombinant plasmid was induced. The purified 4×Aβ15 protein was then obtained after the GST-4×Aβ15 protein was digested by thrombin. Meanwhile, BALB/c mice were used to confirm whether they could generate Aβ specific humoral immunity when immunized with the 4×Aβ15 protein.RESULTS: After identification and DNA sequence analysis, the recombinant plasmid pGEX-4×Aβ15 was constructed. Expression of recombinant 35 kD GST-4×Aβ15 was verified by SDS-PAGE and Western blotting. The 9 kD 4×Aβ15 protein was produced after thrombin digestion of GST-4×Aβ15. Immunizing mice with the 4×Aβ15 protein have induced high titre anti-Aβ antibodies.CONCLUSION:The recombinant prokaryotic vector pGEX-4×Aβ15 was constructed successfully, and the purified 4×Aβ15 protein was obtained after prokaryotic expression. It is demonstrated that the 4×Aβ15 protein is a strong immunogen, confirmed by immunization of BALB/c mice.     

香蕉线条病毒衣壳蛋白功能域基因的原核表达及抗血清制备

 制备香蕉线条病毒广东分离物（BSV-GD）的抗血清,可为BSV-GD 的快速检测和进一步研 究BSV 编码蛋白的功能提供条件。通过常规分子生物学方法,克隆了BSV-GD 衣壳蛋白（Coat protein, CP）功能域基因,构建了该基因的原核表达载体pET28b-CP,并诱导表达了大小约为46.5 kD 的融合蛋 白His-CP。可溶性分析表明该融合蛋白以包涵体形式存在。利用His 标签纯化试剂盒对目的蛋白进行纯 化、回收,获得了高纯度的融合蛋白。以纯化的融合蛋白为抗原免疫健康大耳白兔,成功制备了BSV-GD CP 功能域基因编码蛋白的兔抗血清。Western-blotting 分析表明该抗血清具有很强的特异性,间接ELISA 法检测抗血清效价达204 800 倍以上,对植物材料的合适检测浓度为1︰1 600 ~ 1︰6 400。     

Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

AIM: To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma.METHODS: SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316-SSTR2.pDC316-SSTR2 was cotransfected with rescue plasmid pBHGlox (delta) E1, 3Cre into 293 cells by liposome reagent.Ad-SSTR2 was generated by site-specific recombination and confirmed by PCR.Ad-SSTR2 was propagated in 293 cells and purified.The titer of viral stock was determined by end-point dilution assay.Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan-2 was infected with recombinant adenovirus.RESULTS: pDC316-SSTR2 was successfully constructed.Recombinant adenovirus Ad-SSTR2 was acquired by pDC316-SSTR2 and pBHGlox (delta) E1, 3Cre cotransfected into 293 cells.Ad-SSTR2 was characterized by PCR.The virus titer was 6.0×1012 pfu/L.SSTR2 protein was detected after adenovirus infected capan-2 48 h with Western blotting.CONCLUSION: The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells.This investigation provides the basis for study of gene therapy of pancreatic carcinoma.     

Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.