Cloning of human sperm protein(SP17) and expression in escherichia coli DH5α

AIM: To obtain GST fusion protein of hSP17 gene and construct the recombinant plasmidfor expression in E.coli.METHODS:Total fragment of hS P17 cDNA gene were amplified by RT-PCR,then subcoloned into p GEX-3b to generate recombinant hS P17/pGEX.Right orientation of insert are identified by restricted enzyme digestion.Transform the correct recombinant plasmid into the E.coli DH5a.The expression of fusion proteins hS P17-GST were induced by adding isopropylthiogalactoside(IPTG).RESUL TS and CONCLUSION:The recombinant plasmid hS P17/pGEX-3b could express effectively in E.coli and a high level of fusion protein hsp17-GST with the predicted molecular weight was detected.     

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.     

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.     

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。     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Interaction between human augmenter of liver regeneration and Na+-K+-ATPase in vitro

AIM: To check the physical interaction between GST- Na+-K+-ATPase domain and recombinant human augmenter of liver regeneration (rhALR) by GST pull down assay. METHODS: With PCR and genetic recombinant techniques, the coding region of β subunit of Na+-K+-ATPase was cloned into expressing plasmid pGEX-4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0.1 mmol/L IPTG, the fusion protein GST- Na+-K+-ATPase domain was highly expressed by E.coli DH-5α. After hypersound quassating, the GST- Na+-K+-ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS: Analysis by SDS-PAGE showed the rhALRs of monomer and dimmer in GST- Na+-K+-ATPase domain lane. The Western blotting of the GST-pull down assay showed the same results as well. CONCLUSION: The Na+-K+-ATPase domain is associated with rhALR specifically in vitro.     

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.     

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     

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.     

Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

响应葡萄根瘤蚜侵染的扩展蛋白基因筛选及其多克隆抗体的制备

以对葡萄根瘤蚜敏感的‘克瑞森无核’葡萄(Vitis vinifera ‘Crimson Seedless’)组培苗和抗性砧木‘1103P’组培苗为试材,通过荧光定量PCR技术从扩展蛋白基因家族中筛选到经根瘤蚜侵染后表达上调的扩展蛋白基因家族成员VvEXPA1。采用RT-PCR方法克隆到VvEXPA1基因片段,连接至原核表达载体pET-32a中,转化大肠杆菌DE3菌株,测序确定构建的重组载体pET32a-VvEXPA1开放阅读框正确,并经IPTG诱导表达了融合蛋白。该蛋白以包涵体的形式存在,纯化后免疫新西兰兔,制备扩展蛋白多克隆抗体,通过Western-blot和ELISA检测抗体的特异性及效价。结果显示制备的抗体具有较高的特异性,ELISA显示效价为1:51 200。     

Cloning expression and toxic effect on the host bacterial viability of an outer envelope protein from the strong virulent leptospira lai in China

AIM: Construction of an eukaryote-E. coli shuttle expressing recombinant plasmid which expresses OmpL1 envelope protein of pathogenic Leptospira, serovar Lai strain 017. METHODS: The OmpL 1 gene was amplified by PCR from the leptospiral genome. Then it was cut with restriction enzymes and ligated to the plasmid pBK-CMV. The correct recombinant plasmid was screened out with analysis of restriction enzymes and PCR. After inducing the E. coli baring recombinant plasmid with IPTG,the complete protein of the bacteria was extracted for SDS-PAGE. At the same time, OD600 of the host bacteria was examined at different time after inducing or uninducing with IPTG. RESULTS: Five strains E. coli containing proper recombinant plasmids were screened out. Four strains E. coli expressed a new protein with a weight of 37 kD among them. With the expression of the heterogenous protein,the OD600 of the host bacteria decreased. CONCLUSION: The shuttle expressing plasmid of the OmpL 1 gene of strong virulent Leptospira strain 017 was successfully constructed. Furthermore,the recombinant plasmid expressed the expected OmpL 1 fusion protein in E. coli and the expression of the heterogenous protein had toxic effect on the host bacteria. This work was important for the future research of OmpL1 protein which relates to the diagnosis,new vaccine preparing and the pathogenic mechanism of leptospirosis.     

Expression features of human decorin in E.coli DH5α

AIM: To study the expression features of human decorin in E. coli DH5α.METHODS: The pGEX-4T-1-decorin fusion clone was expressed in E. coli DH5α. Positively expressed clone was selected by SDS-PAGE. The optimized inducing time by 1 mmol/L IPTG was determined. The solubility of GST-decorin fusion protein was analyzed by ultrasonic crush method, and its quality was examined by Western blotting. RESULTS: With the induction of 1 mmol/L isopropy-β-D-thiogalactoside (IPTG), the fusion protein was expressed in E. coli DH5α. The optimized inducing time by 1 mmol/L IPTG was 4 hours. Most of fusion protein existed in the form of inclusion body. The expressed protein was GST fusion protein.CONCLUSION: It is suggested that the fusion protein of GST-decorin may be expressed in E.coli DH5α in a large amount in the form of inclusion body.     

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.     

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.     

Key amino acids of interactions between interferon-induced protein N-Myc interactor and human cytomegalovirus tegument protein UL23

AIM:To investigate the key amino acids of interferon-induced protein N-Myc interactor (Nmi) that interacts with human cytomegalovirus (HCMV) tegument protein UL23. METHODS:According to the previous results, 10 groups of the truncated Nmi fragment were constructed on plasmid pGEX-4T-1. Recombinant plasmids were transformed into E.coli Rosetta (DE3) competent cells, and fusion proteins with GST tag were expressed and purified. The domains on Nmi that mediated the interaction with UL23 were identified by GST-pulldown test. Based on the results of GST-pulldown test, 3 groups of deleted Nmi fragments were inserted into the eukaryotic expression plasmid pcDNA4-Myc by homologous recombination. The plasmid with Flag-tagged UL23 plasmid and wild-type Nmi or deletion mutants were co-transfected into HeLa cells to reconfirm the key amino acids on Nmi that interacted with UL23 by the method of co-immunoprecipitation (Co-IP). RESULTS:The prokaryotic expression vectors of 10 groups of truncated Nmi mutants fused with GST gene were successfully constructed. Three groups eukaryotic expression vectors of Nmi deletion mutants fused with Myc gene were also successfully constructed. The results of GST-pulldown test indicated that the region of 192~202 aa located on Nmi was a key area to interact with UL23. The UL23 binding domain of Nmi in the amino acids 192~202 aa was confirmed by Co-IP, which was consistent with the GST-pulldown results. CONCLUSION:The 192~202 aa of Nmi are key amino acids in the interaction with UL23. This may provide the basis for clarifying the molecular mechanisms by which UL23 plays an important role in the latency of HCMV in host.     

Soluble expression of a CXCL10-loop3-EGF fusion protein and its anti-tumor activity

AIM: To evaluate the implication of CXCL10-loop3-EGF fusion protein for the activities of targeting tumor and anti-angiopoiesis. METHODS: RT-PCR was preformed to amplify CXCL10 coding sequence from PBMC activated by IFN-γ. CXCL10-loop3-EGF fusion gene, which was conducted by Over-Lap Extention PCR, was hinged up with plasmid pTG19-T, transfected to E. coli DH5α and processed positive colony selection. After ligated with plasmid pET32a(+), recombinant CXCL10-loop3-EGF fusion gene was then transfected to E. coli Origami B (DE3) and induced to express its coding fusion protein his-CXCL10-loop3-EGF. The recombinant fusion protein CXCL10-loop3 -EGF was purified by His-bind affinity chromatograph, enterokinase cleavage, ultrafiltration and dislysis. The transwell chemotatic test and HUVEC angiopoiesis inhibition test were performed to determine the anti-tumor responses and anti-angiopoiesis activity of CXCL10-loop3-EGF fusion protein. RESULTS: CXCL10-loop3-EGF fusion protein was successfully constructed and confirmed by SDS-PAGE analysis and Western blotting. Significant PBMC chematatic activity and HUVEC anti-angiopoiesis activity were observed. CONCLUSION: CXCL10-loop3-EGF fusion protein, which has perfect anti-tumor activity, is successfully constructed.     

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。     

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。