Expression and purification of DNA binding domain of NR4A1 / 中南大学学报(医学版)

OBJECTIVE@#To express and purify NR4A1-DNA binding domain (DBD) protein of nuclear receptors.@*METHODS@#The fusion protein PET28a-NR4A1-DBD was constructed and purified with the nickel affinity chromatography, cation-exchange chromatography and gel filtration chromatography.@*RESULTS@#The protein PET28a-NR4A1-DBD was mostly soluable at 24 °C. A total of 2-3 mg/L pure NR4A1 proteins were yielded in bacterial culture and the purity for final fractions of NR4A1-DBD protein were great than 95% by SDS-PAGE analysis.@*CONCLUSION@#Nickel affinity chromatography is effective to purify protein. The protein purity can be further improved by the following methods including cation-exchange chromatography and gel filtration chromatography.

Bioinformatics analysis of differentially expressed proteins in human lung adenocarcinoma / 中国医师杂志

Objective To investigate the biological significance of differentially expressed proteins from human primary lung adenocarcinoma with lymph node metastasis adenocarcinoma (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis,with bioinformatics approach.Methods Cytoscape software was used to analyze a functional enrichment analysis and a protein-protein interaction network from differentially expressed proteins from LNM AdC and non-LNM AdC.Results The top biological processes were related to glucose catabolic process,hexose catabolic process,monosaccharide catabolic process,alcohol catabolic process,and cellular carbohydrate catabolic process.The top molecular functions were related to phospholipase inhibitor activity,lipase inhibitor activity,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,and lipid binding.A protein-protein interaction network of differentially expressed proteins was generated with literature data.Conclusions This bioinformatics analysis demonstrated that glucose catabolic process,alcohol catabolic process,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,ACTB,ANXA1,ANXA2,ANXA3,VCP,NPM1,KRT1,and SUMO4 are significantly associated with a lung adenocarcinoma.These network data provide new insights into the metastasis mechanisms of human lung adenocarcinoma.

Molecular mechanism of SH2B1 in regulating JAK2/IRS2 during obesity development / 中南大学学报(医学版)

Objective In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function.Methods Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. Results SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239~(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1~(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1~(-/-) males than wild-type littermates at 15 weeks of age. SH2B1~(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5~(th) week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates.Conclusion SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.

Screening for nasopharyngeal carcinoma antigens that induce humoral immune response by serological proteomics analysis / 中华检验医学杂志

Objective To identify proteins eliciting a humoral immune response in patients with nasopharyngeal carcinoma (NPC) by a serological proteome analysis, and provide candidate biomarkers for diagnosis and treatment of NPC.Methods Two-dimensional (2-DE) electropboresis was used to separate total cellular proteins from 19 NPC tissues.Separated proteins were transferred onto PVDF membranes and sera from 19 NPC patients and 19 healthy subjects were individually screened by western blotting for antibodies that react against separated proteins.Each tissue samples was subjected to three 2-DE gels and coomassie staining was performed in one of them.The protein spots which selectively reacted with the patient sera were excised from the preparative gels and subjected to further analysis of MOLDI-TOF MS and ESI-QTOF MS/MS.The proteins were identified based on peptide mass fingerprints and peptide sequence tags followed by searching database.Results In this study, 13 NPC associated antigens (HSP 70, HAS,HSP 60, CK 15, LAP 3, α-enolase, EBP 1, CK 19, ribosomal protein P 0, pyrovate dehydrogenase E1,guanine nucleotide-binding protein, prohibitin, Rho-GDI 2) that elicited an antibody response in most of NTC patients were identified.The positivities of these proteins were more than 21% all in NPC patients, but were lower or even absent in normal subjects.Conclusion These 13 NPC associated antigens and their autoantibodies may be useful for NPC diagnosis and treatment.

Methylation and expression of glioma pathogenesis-related protein 1 gene in acute myeloid leukemia / 中南大学学报(医学版)

OBJECTIVE@#To detect the methylation and expression of glioma pathogenesis-related protein 1(GLIPR1) gene in the acute myeloid leukemia (AML) cell lines and bone marrow cells from AML patients, and to determine the relationship between promoter methylation and expression of GLIPR1.@*METHODS@#Five leukemia cell lines, 54 bone marrows from the newly diagnosed AML patients, 48 bone marrows from the acute lymphoblastic leukemia (ALL )patients, 40 bone marrows from the chronic myeloid leukemia (CML) patients,35 bone marrows from control patients, and 8 bone marrows from the complete remission AML patients were collected. RT-PCR and methylation-PCR (MSP) were used to detect the mRNA expression and promoter methylation of GLIPR1, respectively, and the relationship between them was analyzed.@*RESULTS@#The level of GLIPR1 mRNA in the AML cell lines was lower than that in the chronic myeloid leukemia (CML) and ALL cell lines, whereas the methylation level of GLIPR1 in the former was higher than that in the latter. The level of GLIPR1 mRNA in the AML cell lines was significantly increased, but had no obvious changes in the CML and ALL cell lines after 5-aza-2dC treatment. The mRNA level of GLIPR1 in the AML bone marrows (0.38+/-0.20)was obviously lower than that in the ALL bone marrows (0.76+/-0.18), CML bone marrows (0.80+/-0.14), and control bone marrows(0.85+/-0.12). The level of GLIPR1 mRNA in the bone marrows with complete remission AML was obviously higher than that in the AML bone marrows before the treatment (0.78+/-0.13 vs. 0.36+/-0.20); but there was no obvious difference between the ALL bone marrows and the control bone marrows, and the CML bone marrows and the control bone marrows (both P>0.05). The positive rate of GLIPR1 gene methylation in the AML bone marrows (81.5%) was obviously higher than that in the ALL bone marrows(37.5%), CML bone marrows (27.5%) and the control bone marrows(14.3%). The positive rate of GLIPR1 gene in the bone marrows with complete remission AML was obviously lower than that in the bone marrows before the treatment (12.5% vs. 75.0%), but there was no obvious difference between the ALL bone marrows and between the control bone marrows,and the CML bone marrows and the control bone marrows (both P>0.05). There was a negative correlation between the mRNA level and methylation status of GLIPR1 in the AML bone marrows.@*CONCLUSION@#GLIPR1 expression is downregulated or even lost by promoter methylation in AML, and the expression and methylation level of GLIPR1 gene may have some significance in evaluating the curative effect of AML patients.

Establishment of protein expression profile of laser capture microdissection-purified nasopharyngeal carcinoma tissue / 中南大学学报(医学版)

OBJECTIVE@#To establish the protein expression map of nasopharyngeal carcinoma (NPC), and provide a basis for proteomic study of NPC.@*METHODS@#Laser capture microdissection (LCM) was used to isolate cancer cells from NPC tissues. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of LCM purified NPC cells. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins, and bioinformatics was used to construct the 2-DE database of NPC proteome.@*RESULTS@#A 2-DE reference map of NPC was established. On the 2-DE map, a total of (1 312+/-30) protein spots were detected, and 427 protein spots representing 241 non-redundant proteins were identified. The 2-DE database of NPC proteome was constructed. These data could be accessed at our website (http://www.xyproteomics.org).@*CONCLUSION@#A protein expression profile of LCM purified NPC tissues has been established for the first time, which provides useful information and source for proteomic study of NPC.

Comparative proteome analysis of laser capture microdissection for purified primary tumor and lymph node metastatic tumor in human lung squamous carcinoma / 中南大学学报(医学版)

Objective To search for lymph node metastasis-associated proteins in human lung squamous carcinoma (hLSC).Methods Laser capture microdissection (LCM) was used to purify the target cells from lung primary tumor and matched lymph node metastatic tumor in hLSC. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected tumor cells from lung primary tumor and matched lymph node metastatic tumor. PDQuest software was applied to analyze 2-DE images. Differential protein spots between the two types of tissues were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The expression of Rho-GDIα, one of the differential proteins, in the microdissected lung primary tumor cells (LPTC) and matched lymph node metastatic tumor cells (LNMTC) was detected by Western blot. Results In the present study, 2-DE patterns of microdissected LPTC and LNMTC were established, and 22 differential proteins in the above two tissues were identified, of which 14 were down-regulated in LNMTC and 8 were up-regulated in LNMTC.Conclusion The 22 differential proteins may play some roles in the process of lymph node metastasis in hLSC, and the data provide new clues for metastasis-associated biomarker screen and mechanism of hLSC.

Molecular mechanism of SH2-Bβ in regulating JAK2/STAT3 during development of obesity / 中华内分泌代谢杂志

The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.

14-3-3 sigma Promoter de-methylation and gene expression in nasopharyngeal carcinoma cell lines / 国际肿瘤学杂志

Objective To investigate the methylation status of 14-3-3σ promoter in nasopharyngeal carcinoma cell lines and the influence of de-methylation treatment on 14-3-3σ expression. Methods Methylation status of 14-3-3σ gene promoter and 14-3-3σ mRNA expression were detected by methylation specific PCR (MSP) and RT-PCR in nasopharyngeal carcinoma cell lines CNE1, CNE2,5-8F,6-10B and immortalized nonneoplastic human nasopharyngeal epithelial cell line, NP69. Four nasopharyngeal carcinoma cell lines were treated with 5-asa-2' -deoxycytidine(5-aza-2dC) in different concentration for 72 h, then 14-3-3σ promoter meth-ylation status and m RNA expression were assessed, and western-blot was performed to detect the expression of 14-3-3σ protein. Results 14-3-3σ promoter methylation was detected by MSP in all of the four nasopharyn-geal carcinoma cell lines untreated by 5-aza-2dC whereas not in the treated ones or the immortalized human na-sopharyngeal epithelial cell line, NP69. Accordingly, 14-3-3σ mRNA expression was significantly discounted in untreated nasopharyngeal carcinoma cell lines as compared with NP69. 5-aza-2dC treatment dose-depend-ently reversed 14-3-3σ promoter methylation status and consequently upregulated the expression of 14-3-3σmRNA and protein in 4 nasopharyngeal carcinoma cell lines. High-differentiated CNE1 was more sensitive to 5-aza-2dC than lowly-differentiated CNE2, 5-8F and 6-10B. Conclusion Promoter methylation directly leads to decreased 14-3-3σ gene expression in nasopharyngeal carcinoma cell lines, and 14-3-3σ promoter de-methylation perhaps indicates a new target for nasopharyngeal carcinoma treatment.

Differentially expressed proteins of the fibroblast cell-secreted protein in nasopharyngeal carcinoma / 中华检验医学杂志

Objectives To establish the two-dimensional electrophoresis(2-DE)profile of cell. Secreted proteins.Difierential expression profiling of fibroblast cell secreted proteins between nasopharyngeal carcinoma and normal nasopharyngeal tissue was analyzed.Methods Five tissue specimens each from patients with nasopharyngeal carcinoma and nasal polyp were collected individually.Fibroblast eells from above-mentioned tissue were cultured in serum-free medium,and cell-secreted proteins from the cultured medium were harvested by uhrafihration concentration and desalination.Samples were analyzed by 2-DE,and the differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Galectin-1 wa8 analyzed by EUSA test.Results 2-DE diagram of fibroblast cell-secreted proteins Was constructed.1 8 protein spots displayed quantitative changes in expression,and 11 protein spots among them were identified by mass speetrometrv.3 proteins including cystatin C,complement subcomponent C1S precursor,heterogeneous nuclear ribonueleoprotein A1 were down-regulated in the cultured medium of nasopharyngeal carcinoma associated fibroblast cells(CAFs). Nevertheless,the rest cell-secreted proteins including galectin-1,14-3-3 protein sigma,eathepsin L and etc,were up-regulated.Meanwhile,the expression of galectin-1 in the cultured medium was also analyzed and Its results were compared between CAFs and the normal fibroblast cells by ELISA.There Was statistical significance difference between them,and galectin-1 was up-regulated in the cIlltured medium of CAFs.Conclusions The changes of fibroblast cell-secreted proteins during nasopharyngeal carcinogenesi8 are analyzed by 2-DE analysis.The variation of pattern of secreted proteins is involved in signal transduction,protein synthesis,degradation and other pathways.CAFs may regulate tumor microenvironment by the abeve-mentioned pathways,and influence nasopharyngeal carcinogenesis,progress,invasion and metastasis.This study provided experimental basis for the eell secreted proteomics studv in future.

Quantitative Proteomics Analysis of LCM Purified Stroma of Nasopharyngeal Carcinoma and Normal Nasopharyngeal Mucosa / 生物化学与生物物理进展

The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.

Characterization of Key Regulatory Elements of LCRG1 Promoter / 生物化学与生物物理进展

LCRG1(laryngeal carcinoma related gene1,LCRG1),a new candidate tumor suppressor gene of laryngeal carcinoma.However,it is known little about the possible regulatory mechanisms of LCRG1 gene expression.Restriction endonuclease digestion was used to obtain a set of the 5',or 3'deletion mutants from the region(-169～+127) of the LCRG1 gene.It has been found that the minimal promoter of the LCRG1 gene is mapped at the region from-169～-57.Linker scanning mutational analysis in the region(-169～+127) of the LCRG1 gene was used to identify the crucial cis-elements within the promoter region,The key cis-elements are within the region from-137～-122.SP1,E2F1/DP1,EKLF and ZF9 transcription factor binding site sites were predicted in the region by bioinformatics analysis.Co-transfection with each of a panel of the expression plasmids of the known transcription factors with the relevant reporter construct indicates Sp1 is potent transcription factor for enhancement of the promoter activity,SP1 can also up-regulate the endogenous expression of LCRG1 gene.Electrophoretic mobility shift assay(EMSA) was applied to verify that the key cis-elements of LCRG1 gene exist sequence of Sp1 binding sites.The findings,which showed that the key cis-elements within the region from 137～-122 play an important role in expression of the LCRG1 gene,provide a novel evidence for further study of the function of LCRG1 gene.

Cloning and Identification of Promoter of Human LCRG1 Gene / 生物化学与生物物理进展

Laryngeal carcinoma related gene 1(LCRG1) is a candidate tumor suppressor gene of Laryngeal carcinoma. To further investigate its transcriptional regulation, the transcriptional start sites for LCRG1 gene have been identified by 5′ RACE (rapid amplification of cDNA ends) based on the bioinformation analysis of LCRG1. Then eleven luciferase expression vectors which contained potential human LCRG1 gene promoter were constructed. Luciferase reporter assay indicated that LCRG1 promoter region was mainly located in -169～+127 region nearby the major transcriptional start site. These results suggested that the region (-169～+127) includes an essential promoter for human LCRG1 gene transcription.

Comparative Proteomics Analysis of LCM Purified Nasopharyngeal Carcinoma and Normal Nasopharyngeal Epithelial Tissue / 生物化学与生物物理进展

To search for nasopharyngeal carcinoma (NPC) biomarkers,laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected NPC and NNEC,PDQuest software was applied to analyze 2-DE images,and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. The expression of cytokeratin 8(CK8),one of the differential proteins,in the microdissected NPC and NNEC as well as 4 NPC cell lines with different differentiated degrees and/or metastatic potentials was detected by Western blot. Immunohistochemistry was also used to detect the expression of CK8 in paraffin-embedded tissues including 63 cases of primary NPC,28 cases of NNET and 20 cases of cervical lymphonode metastasis. In the present study,2-DE patterns of microdissected NPC and NNEC were established,and 29 differential proteins in the above two tissues were identified,of which 15 only expressed or up-regulated in NPC and 14 only expressed or up-regulated in NNET. The expression level of differential protein CK8 between the NPC and NNET was selectively confirmed,and was found to be related to the differentiation and/or metastasis of NPC cell lines. Significant down-regulation of CK8 was observed in NPC compared with NNET,and significant up-regulation of CK8 was also observed in lymphonode metastasis compared with primary NPC. The data suggest that CK8 may be related to the differentiation and lymphonode metastasis of NPC,and may serve as molecular biomarkers for metastasis and differentiation of NPC.

Differential Proteomic Analysis of Sera From Lung Squamous Carcinoma Patients and Healthy Individuals / 生物化学与生物物理进展

To screen for serum biomarkers for lung squamous carcinoma, two-dimensional gel electrophoresis (2-DE) was performed to separate serum proteins from healthy individuals and stage 1 lung squamous carcinoma(LSC) patients, respectively. PDquest software was used to analyze 2-DE images, and the differential serum protein spots between the healthy individuals and LSC patients were identified by ESI-Q-TOF MS/MS. Then Western blot and immunohistochemistry were used to detect the expression levels of haptoglobin-2(HP-2), one of the differential proteins, in the sera and tumor tissues in the patients with LSC, respectively. 2-DE maps of serum proteins from healthy individuals and stage 1 LSC patients were established. Ten differential serum protein spots were detected, four proteins of which were identified by MS/MS. Western blot showed that the serum level of HP-2 in the LSC patients was significantly higher than that in healthy individuals, but was not associated with LSC staging. Immunohistochemistry showed that the expression level of HP-2 in the LSC tissues was significantly higher than that in the normal bronchial epithelial tissues adjacent to tumors. The results indicated that serum HP-2 protein is a candidate biomarker for LSC, and might be useful for diagnosis of LSC. Up-regulation of HP-2 in the LSC tissues may contribute to the high serum level of HP-2 in the patients.

Identification of Proteins Associated With p53 in Nasopharyngeal Carcinoma Cell Line CNE2 by Proteomics and RNAi Techniques / 生物化学与生物物理进展

In order to elucidate the mechanisms of p53 overexpression in nasopharyngeal carcinoma (NPC) and detect proteins associated with the function of p53 in high throughout screening, p53 which knockdown human NPC CNE2 cell line (CNE2sip53) were successfully established by using stable RNA interference (RNAi). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of CNE2sip53 and its control cell line CNE2/pSUPER, and PDQuest software was applied to analyze 2-DE images. Twenty-two differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3? etc.) , and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, the differential expression levels of the partial proteins (HSP27, 14-3-3?, GRP75) were confirmed by Western blot analysis and compared with CNE2 and CNE2 cells transfected with pcDNA3.1-FLAG, CNE2 cells transfected with pcDNA3.1-FLAG-p53 had obvious down-regulations of HSP27 and 14-3-3?, and an up-regulation of GRP75. The 22 differentially expressed proteins could be divided into five groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence,cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of p53 in NPC, and will be valuable for further to study the mechanisms of p53 overexpression and inactivation in NPC.

Comparative Proteomics Analysis of Normal Colonic Epithelium in Young and Old People / 生物化学与生物物理进展

The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the molecular mechanisms of the aging and susceptibility of aged colonic epithelium to carcinogenesis is still unclear. Identification of aging related proteins in colonic epithelium will help to reveal the molecular mechanisms of colonic epithelial aging and age-related colonic diseases. Therefore, the total proteins of human normal colonic epithelial tissues from 10 young and 10 old men were separated by two-dimensional gel electrophoresis(2-DE), respectively. PDQuest software was applied to analyze 2-DE images, the differentially expressed protein spots of colonic epithelium between young and old groups were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), and the expression levels of partial identified proteins were determined by real-time quantitative PCR and immunohistochemistry. Well-resolved, reproducible 2-DE maps of human colonic epithelial tissues from young and old men were established, 17 aging related proteins were identified by MALDI-TOF-MS, and the differential expression levels of partial identified proteins were confirmed by real-time quantitative PCR and immunohistochemistry. The results indicate that injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium, and four differential proteins (guanine nucleotide-binding protein beta subunit-like protein, stress-70 protein, 40 S ribosomal protein SA and chloride intracellular channel protein1) may be involved in susceptibility of aged colonic epithelium to carcinogenesis.

Screening of EGFR-regulated Secreted Proteins in Human NPC Cell Line CNE2 / 生物化学与生物物理进展

In order to screen EGFR-regulated secreted proteins in human nasopharyngeal carcinoma(NPC), and to reveal the role and mechanism of epidermal growth factor receptor(EGFR) in the pathogenesis of NPC. NPC cell line CNE2 cells were cultured in serum-free medium and stimulated by transforming growth factor-? (TGF-?) for 24 h in experimental group. Control CNE2 cells were cultured at the same condition but without TGF-? stimulation. The culture medium of control and experimental cells was desalted and concentrated through ultrafiltration to prepared the total secreted proteins. Two-dimensional gel electrophoresis (2-DE) was used to separate the secreted proteins of control and experimental cells, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the control and experimental cells were identified by desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The 2-DE patterns of the secreted proteins of TGF-? stimulated and un-stimulated CNE2 cells were established, 22 differential proteins spots between the two groups of cells were found, and 8 non-redundant proteins were identified with MALDI-TOF-MS, the functions of which were involved in invasion, metastasis, apoptosis and proliferation of cancer cells. The data will be valuable for further to study the role and mechanism of EGFR in the pathogenesis of NPC.

Recombination of human HER2 gene encoding the extracellular domain and purification of fusion protein / 生物医学工程学杂志

To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature (30 degrees C) and higher optical density (A600= 1.8) with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein, and thus have laid a foundation for further work on functional study and antibody preparation for HER2.

Cloning, expression and purification of rabbit metallothionein-I gene in Escherichia coli / 生物医学工程学杂志

The cDNA encoding the rabbit metallothionein-I was amplified by RT-PCR from the rabbit liver induced by cadmium and cloned into prokaryotic fusion expression vector pQE40. Then it was transformed into Escherichia coli M15. Positive expression clones were detected by colony blotting. Target protein solubility was determined by Western blotting analysis. The optimal induction condition of the level of protein expression with IPTG induction was established by SDS-PAGE electrophoresis and ImageMaster VDS software analysis. The fusion protein can be purified from lysates with Ni-NTA agarose. We found that the fusion protein with apparent molecular weight 32 KD existed in two ways: soluble and insoluble in Escherichia coli. After 1 mM IPTG induction, the level of expression of the fusion protein increased with the prolongation of induction time and reached a peak in 9 h by ImageMaster VDS software analysis, accounting for 57.4% of all the insoluble protein. The purified fusion protein was obtained by Ni-NTA affinity chromatography. This fusion protein can be used in further studies on the preparation of MT-I protein and development of protein product.