ATPase inhibitory factor 1-a host cell protein molecule interacting with human cytomegalovirus pUL23 protein

AIM: pUL23, the product of human cytomegalovirus (HCMV) gene UL23 was identified as one of tegument proteins. The aim of this research is to investigate the function of pUL23 during HCMV life cycle.METHODS: GAL4 yeast two-hybrid assay was performed to screen the human fetal kidney cDNA library to obtain host cell protein molecules which interact with pUL23 of HCMV. Then the GST-pulldown experiment was applied to confirm the protein interactions identified by yeast two-hybrid.RESULTS: ATPase inhibitory factor 1 (ATIF1) was selected from host cells using yeast two-hybrid assay. GST-pulldown experiments in vitro further confirmed the interaction between ATIF1 and pUL23.CONCLUSION: pUL23 of HCMV can interact with ATIF1 in host cell, which may provide the evidence for understanding the function of pUL23 in the life cycle of HCMV.     

Studies on the human cytomegalovirus protein pUL23 by establishment of HELF cell line stably expressing pUL23 gene

AIM: To establish HELF cell line with stable and effective expression of human cytomegalovirus (HCMV) UL23 gene as a powerful tool to study the situation of viral proteins in host cells in vitro. METHODS: UL23 gene was amplified by PCR from HCMV and then recombinant plasmid pLEGFP-N1-FLAG-UL23 was constructed by molecular cloning techniques. Artificial retroviral viruses containing HCMV UL23 gene were obtained by transferring plasmid pLEGFP-N1-FLAG-UL23 into the AmphoPackTM-293 package cells. The HELF cells were infected by the artificial retroviral particles. The positive HELF cells which stably expressed the UL23 were obtained by G418 selection. The location of the viral proteins in the cells was observed by laser confocal microscope. RESULTS: UL23 gene was amplified by RT-PCR from the positive HELF cells, and the UL23 fusion protein was expressed correctly and confirmed by Western blotting in the positive cells, indicating that the UL23 gene was stably integrated into the chromosome of HELF cells. The viral UL23 protein expressed in HELF cells was located in the cytoplasm observed by means of laser confocal microscope. CONCLUSION: Transgene cell strain stably carrying UL23 gene is successfully constructed by retroviral gene transfer techniques. The location of HCMV pUL23 in the host cells implicates that this vial protein exerts its function in cytoplasm.     

Subcellular localization of ZNF580-EGFP fusion protein

AIM: To construct a eukaryotic expression plasmid for enhanced green fluorescent protein (EGFP) and ZNF580 fusion protein, and study its subcellular localization in the transfected MGC803 cells. METHODS: The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame (1-172aa), ZNF580 amino terminus (1-93aa) and ZNF580 carboxyl terminus (94-172aa). The three cDNA segments of PCR were cloned into pGEM-T vector. Then they were subcloned respectively into plasmid pEGFP-C1 (enhanced green fluorescent protein). The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy. RESULTS: Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580 (1-172), pEGFP-ZNF580 (1-93) and pEGFP-ZNF580 (94-172) transgenic plasmid were correct. The fusion proteins of EGFP-ZNF580 (1-172) and pEGFP-ZNF580 (94-172) were localized in the nuclei. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed. The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus (C2H2 zinc finger domain).     

芥菜开花调控因子SVP 与FLC 蛋白互作的结构域筛选与鉴定

 为阐明芥菜开花路径核心调节子SVP与FLC相互作用的结构域,从酵母重组质粒pGADT7SVP、pGBKT7FLC分别亚克隆了5个SVP截短体（SVP1 ~ 5）和5个FLC截短体（FLC1 ~ 5）。SVP1 ~ 5与FLC1 ~5编码蛋白的结构域均分别为MI、MIK、K、IKC和KC。利用酵母双杂交体系,分别构建酵母猎物质粒pGADT7SVP1 ~ 5与诱饵质粒pGBKT7FLC1 ~ 5,并转化对应的酵母Y187、Y2HGold菌。酵母转化子Y187［pGADT7SVP2 ~ 5］能与Y2HGold［pGBKT7FLC］融合,并可在选择性固体培养基QDO/X/A上长出蓝色菌落,表明FLC能与截短体蛋白SVP2 ~ 5异源结合,SVP的K域（SVP3）可独立作用于FLC蛋白。此外,Y187［pGADT7SVP］× Y2HGold［pGBKT7FLC2 ~ 5］也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,表明FLC的K域（FLC3）也可独立作用于SVP。进一步研究发现：Y187［pGADT7SVP3］× Y2HGold［pGBKT7FLC3］正向杂交以及Y187［pGADT7FLC3］× Y2HGold［pGBKT7SVP3］载体互换后杂交均可相互作用,表明SVP的K域（SVP第96 ~ 173位氨基酸区域）与FLC的K域（FLC第114 ~ 167位氨基酸区域）能够异源结合,是介导SVP与FLC蛋白互作的关键结构域。     

Identification of oncogene TRE17 interaction with troponin C2

AIM: To identify the proteins bound to the product of oncogene TRE17. METHODS: Bait plasmids TRE17/pGBKT7 and TRE17(447)/pGBKT7 were constructed in this study and used in yeast two-hybrid to screen human skeletal muscle cDNA library for binding partners of TRE17. The interaction of TRE17 with its partner was tested by GST-pulldown assay. RESULTS: TRE17 associated with 6 proteins, including pur alpha, myosin light chain 2, lactate dehydrogenase A, and troponin C2, as demonstrated by yeast two-hybrid. Among them, only troponin C2 interacted with both TRE17 and TRE17(447). GST-pulldown assay confirmed the association of TRE17 with troponin C2, not GST protein. CONCLUSION: Troponin C2 specifically associates with TRE17 oncogene, which provides some new clues to make more researches on mechanism of TRE17 oncogene's transformation.     

Identification of domain of protein kinase R interacting with hepatitis C virus core protein

AIM: To observe if hepatitis C virus (HCV) core protein (CP) influences the expression level of protein kinase R (PKR) and to map the direct interaction domain between PKR and CP.METHODS: The expression levels of PKR in Huh-7,Huh-7 transfected with CP plasmid and replicon Huh-7 harboring selecting full length of HCV genome were studied.HCV structure and non-structure proteins in replicon Huh-7 with interferon (IFN) stimulation were compared.Co-immunoprecipitation and glutathione S-transferase (GST) binding assay were done between PKR and CP.RESULTS: PKR expression level in replicon Huh-7 was higher than that in Huh-7 and Huh-7 transfected with CP expression plasmid.PKR was increased but structure and non-structure proteins in replicon Huh-7 were decreased after treated with IFN.The N-terminal 1-180 amino acid of PKR was the key binding site to CP.CONCLUSION: CP directly binds to N-terminal 1-180 amino acid of PKR and leads to constitutive expression of PKR,which interferes signal transfer mediated by PKR.The interaction between CP and PKR might be a novel model of virus protein-cell protein interaction,which might play an important role in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.     

Specific cytotoxicity of TCR Vα23 - Vβ13 gene-modified T-cells

AIM: To analyze the cytotoxicity of TCR Vα23 - Vβ13 gene-modified T-cells specific to diffuse large B-cell lymphoma(DLBCL) associated antigens in vitro. METHODS: The identified full-length TCR Vβ13 and Vα23 genes of monoclonally expanded T-cells in the peripheral blood from a DLBCL patient were amplified and cloned. The bicistronic recombinant plasmid TCR Vβ13 -IRES-TCR Vα23 was constructed and transfected into T-cells isolated from a healthy individual by Nucleofector^TM technology. The mRNA expression of antigen-specific TCR Vα23 and Vβ13 genes and the corresponding proteins were determined by real-time PCR and Western blotting, respectively. The specific cytotoxicity of TCR gene-transferred T-cells in vitro was detected. RESULTS: The recombinant plasmid was constructed successfully and verified by restriction enzyme digestion and nucleotide sequencing. The co-expression of antigen-specific TCR Vα23 and Vβ13 genes at mRNA and protein levels in the transfected healthy T-cells were observed in vitro. Three days after transfection, the cytotoxicity of TCR gene-transduced CD3⁺T-cells against Toledo cells was significantly higher than that against Raji cells or Molt-4 cells. The DLBCL-specific cytotoxic T-lymphocytes(CTL) were inducted. CONCLUSION: The bicistronic eukaryotic expression plasmid TCR Vβ13 -IRES-TCR Vα23 specific to DLBCL-associated antigens is constructed successfully. The TCR gene-transferred T-cells display the ability of DLBCL-specific cytotoxicity.     

Construction of human HSV-1 UL40 gene recombinant and its application in siRNA screening

AIM: To construct the recombinant of human herpesvirus Ⅰ(HSV-1) UL40 gene and to screen siRNAs of silencing efficiently human HSV-1 UL40 gene expression.METHODS: The recombinant UL40-EGFP plasmid (pEGFP-N1-UL40) was constructed by cloning the UL40 gene into pEGFP-N1.siRNA target UL40 gene and pEGFP-N1-UL40 were cotransfected into Vero cells.The effects of RNAi were detected by green fluorescence signals.RESULTS: siRNA3,siRNA4 reduced prominently the expression of UL40 gene.The silence efficiency was 76.99% and 84.00% respectively.CONCLUSION: We succeed in construction of the pEGFP-N1-UL40 recombinant,and screen out siRNA3 and siRNA4 of silencing efficiently human HSV-1 UL40 gene expression.     

不同氮素水平对草莓氨基酸和蛋白质的影响

对不同氮素水平下草莓(FragariaananssaDuch.)果实中的氨基酸和蛋白质进行了分析,结果表明:在所测出的17种氨基酸中,天冬氨酸、谷氨酸、丙氨酸、亮氨酸等是草莓主要的氨基酸,其中天冬氨酸含量最高,占总氨基酸的21.27%～26.83%,其含量与总氨基酸之间存在显著相关性,回归方程为yaa=4.2545+1.6763xAsp(r=0.6858),并且施氮量对这些主要氨基酸的含量变化幅度影响较大。花期追施不同水平氮肥后,随着施氮量增加,蛋白质和氨基酸含量呈增加趋势,必需氨基酸含量亦增加,但占总氨基酸的比例下降。不同氮素水平(低量、中量、高量)下,蛋白质与氨基酸的含量均是随成熟期先下降后上升,而对照(不施氮肥)呈一直下降趋势。蛋白质与氨基酸之间也存在相关性,回归方程为yPr=1.2665+3.4259xaa(r=0.7664)。     

Expression of GST fusion proteins of human cytochrome P2B6 and preparation of anti-cytochrome P2B6 polyclonal antibody

AIM: To get large amounts of pure antigens to raise specific antibodies and to perform quantifications.METHODS: CYP2B6 (cytochrome P) cDNA fragments was ligated into BamHI restricted PGEX-3b to generate recombinants PGEX/2B6. We identified recombinants PGEX/2B6 by EcoRI digestion. The expression of fusion proteins were induced by adding isopropyl-thiogalactoside(IPTG). Several clones showed high-level expression of fusion proteins. Insoluble proteins was isolated from the bacteria and the fusion proteins was recovered and purified from a preparative (2mm) SDS-PAGE. The polyarcrylamide gel containing the fusion proteins glutathione S-transferase(GST-2B6) were used to immunize BALB/C mice from which polyclonal ascites fluid was prepared. The purified fusion proteins GST-1A1(GST fusion protein of CYP1A1 cDNA246~386aa expressed in this library, purified by preparative SDS-PAGE), GST-2B6 were used to test the specificity of 2B6pAb. RESULTS:Fusion proteins constructed between GST and CYP2B6 was expressed in Escherichia coli DH5α. Mouse antibodies are raised against the fusion proteins GST-2B6. 2B6pAb was fond to be specific antibody.CONCLUSION:Recombinant PGEX/2B6 were constructed and purified fusion proteins GST-2B6, and specific 2B6pAb were obtained.     

Cloning,expression and protein structure prediction of a novel SCF-TPO fusion gene

AIM: To obtain the high expression of recombinant human stem cell factor-thrombopoitin (SCF-TPO) fusion gene and predict its structure property. METHODS: Four primers were designed according to known sequence of TPO and SCF to amplify the functional amino acid domain of TPO and SCF by RT-PCR, respectively from fetus hepatocytes. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in BL21(DE3)plysS. The fusion protein property, such as second structure, flexibility,and hydrophilicity were predicted by DS Gene and Protscale software. RESULTS: The expression vector, pET32a/SCF-TPO was constructed and the high expression of the SCF/TPO fusion protein was obtained, with the expression amount of up to 40% of the total cellular protein. DS Gene1.5 and Protscale predict no new antigenicity in fusion protein, and the second structure and ioelectric point have no changes except four amino acids change in first structure. There are high flexibility and low hydrophilicity in the linker peptide. CONCLUSION: High expression of SCF-TPO fusion protein has been obtained and protein prediction shows that the fusion protein design is reasonable, which lay foundation for further study of biological fundation of SCF-TPO fusion protein.     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.     

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.     

利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究

选择葡萄八氢番茄红素脱氢酶(Phytoene desaturase)基因VviPDS1为靶标,利用CRISPR/Cas9系统构建基因敲除载体,瞬时转化葡萄叶片原生质体,检测到不同类型的突变。通过农杆菌介导转化‘无核白’葡萄胚性愈伤组织,筛选获得卡那霉素抗性植株71株。经PCR鉴定,其中53株为阳性植株,阳性率为74.64%。测序结果表明,共有20株在靶点发生不同类型的突变,编辑效率为37.74%;其中9株产生了双等位基因突变。对其进行氨基酸序列预测,在第202位氨基酸之后发生了不同程度的变异。利用CRISPR/Cas9系统敲除VviPDS1获得的突变体植株呈现整体矮化,其叶片出现不同程度白化。表明CRISPR/Cas9系统可以通过细胞中的瞬时或稳定表达进行基因编辑,可以实现在葡萄编辑植株中产生纯合敲除。     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

甜菜蔗糖磷酸合成酶基因的克隆及序列分析

甜菜蔗糖磷酸合成酶（Beta vulgaris sucrose phosphate synthase,BvSPS）是甜菜体内控制蔗糖合成的关键酶之一。根据Genbank上的BvSPS序列（Genbank accession no.X X81975）,利用RT-PCR方法,从甜菜体内扩增获得了BvSPS基因的cDNA全长,并将其连接到pMD 18-T载体上。通过利用PstI、XbaI和EcoRI对重组质粒pMD 18-T-BvSPS进行酶切鉴定,进一步确定了重组质粒的正确性。利用生物信息学软件分析结果表明,该基因cDNA全长为3138bp,编码1045个氨基酸,蛋白质分子量为118.18kDa,等电点为6.15,该蛋白被预测定位于细胞核内。     

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.     

Effect of somatostatin analogue octreotide on metabolism of LPS-induced A549 cells

AIM: To investigate the effects of octreotide on metabolism in the A549 cells treated with lipopolysaccharides (LPS). METHODS: The technologies of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were used to test the metabolism of lung A549 cells subject to different treatment with LPS and/or octreotide. The results were visualized and checked by chromatogram, and the corresponding intensity data were analyzed by the principal component analysis(PCA)method. The metabolites with different expression and the underlying interaction network were resolved. RESULTS: The metabolism analysis by LC/MS method indicated that there were different expression levels between different treated groups. Further analysis was carried out by orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and the different expressed metabolites were obtained, which were mainly amino acids and phospholipids. By analyzing with GC/MS method and t-test, the different expressed metabolites were mainly organic acid, saccharides and amino acid metabolite. The interaction network diagram was constructed about the response of A549 cells induced by LPS and/or octreotide, including glycolysis/gluconenogenesis, tryptophan metabolism, galactose metabolism, urine cycle and citrate cycle. Fourteen key components were found such as serotonin, indole, threonine, serine, glucose, phenylalanine, lactose, fumarate, 4-hydroxyphenyllactate, aspartate, asparagine, putrescine, proline and succinate. CONCLUSION: In octreotide treated LPS-induced A549 cells, the main metabolites are organic acid, saccharides, amino acids and phospholipids. The interaction network is constructed, including 5 metabolic pathways and 14 key components.     

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.