ABSTRACT
In order to investigate the expression of heavy chain of HLA-B * 2705 in prokaryotic system and identify its activity, the extra-membrane gene fragment of HLA-B * 2705 was amplified from full-length HLA-B*2705 cDNA by PCR and cloned into pGEM-T vector. After identification by sequencing, the prokaryotic expressing vector pET32a (+)-B * 2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLA-B * 2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLA-B * 2705 fusion protein are obtained as basis for the further studies.
Subject(s)
Humans , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , HLA-B27 Antigen , Classification , Genetics , Allergy and Immunology , Metabolism , Immunoglobulin Heavy Chains , Genetics , Allergy and Immunology , MetabolismABSTRACT
This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.
Subject(s)
Humans , B7-2 Antigen , Genetics , Bacterial Proteins , Genetics , CD28 Antigens , Allergy and Immunology , Cloning, Molecular , Escherichia coli , Genetics , Gene Expression , Recombinant Fusion Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of oncoprotein c-erbB2 in primary breast cancer and to analyze its relation to its prognosis.</p><p><b>METHODS</b>Immunohistochemical staining for c-erbB2 was performed on paraffin-embedded specimens of primary breast cancer from 284 patients, and the relation to its prognosis was statistically analyzed.</p><p><b>RESULTS</b>Positive expression rate of c-erbB2 was 26.8% (76/284) in 284 primary breast cancer patients. Expression of c-erbB2 was positively correlated with the status of lymph node metastasis (P = 0.003). Univariate analysis indicated that c-erbB2 expression is a significant prognostic factor for the disease-free survival (DFS) (P = 0.024) and overall survival (OS) (P = 0.002), while multivariate analysis demonstrated that c-erbB2 is an independent prognostic factor for OS (P = 0.023). Moreover, tumors with c-erbB2 positive expression are more tend to metastasis to other viscera than those with c-erbB2 negative. c-erbB2 expression has different prognostic values for patients with different status of estrogen receptor (ER) and lymph node metastasis.</p><p><b>CONCLUSION</b>c-erbB2 expression is an independent prognostic factor for total survival time in primary breast cancer patients, and its prognostic values are different according to the different ER status and lymph node metastasis.</p>
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Breast Neoplasms , Metabolism , Pathology , Therapeutics , Carcinoma, Ductal, Breast , Metabolism , Pathology , Therapeutics , Chemotherapy, Adjuvant , Disease-Free Survival , Follow-Up Studies , Lymphatic Metastasis , Mastectomy, Radical , Prognosis , Proportional Hazards Models , Radiotherapy, Adjuvant , Receptor, ErbB-2 , Metabolism , Receptors, Estrogen , Metabolism , Survival RateABSTRACT
<p><b>OBJECTIVE</b>To study the expression of recombinant human SCF-TPO fusion protein and its biological function.</p><p><b>METHODS</b>Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography.</p><p><b>RESULTS</b>The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml.</p><p><b>CONCLUSIONS</b>Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.</p>
Subject(s)
Humans , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Cell Survival , Genetics , Physiology , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Genetic Vectors , Recombinant Fusion Proteins , Genetics , Metabolism , Physiology , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Factor , Genetics , Metabolism , Physiology , Thrombopoietin , Genetics , Metabolism , PhysiologyABSTRACT
<p><b>AIM</b>Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin (T-T) with more stability, efficiency, and lower toxicity.</p><p><b>METHODS</b>The expression vectors of TPO and T-T, pET32 a(+)/TPO and pET32 a (+)/T-T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software.</p><p><b>RESULTS</b>The expressing vectors pET32a(+)/TPO and T-T were constituted correctly and expressed in origami (DE3), and their expression efficiency were more than 40 percent of total protein. T-T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T-T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T-T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and beta-sheet structure.</p><p><b>CONCLUSION</b>We have constructed correctly and expressed T-T with high level in E Coli. Protein characteristics prediction of T-T accords with our design.</p>
Subject(s)
Cloning, Molecular , Escherichia coli , Gene Expression , Genetic Vectors , Recombinant Fusion Proteins , Genetics , Thrombopoietin , GeneticsABSTRACT
The objective was to identify some biochemical and physical properties for fusion protein IL6D24-PE40KDEL. Edman degradation, SDS-PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N-terminus is Met-Ile-Asp-Lys-Gln-Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24-PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS-PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24-PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24-PE40KDEL is a new targeting protein with bioactivity of specific killing effect.
Subject(s)
Humans , ADP Ribose Transferases , Chemistry , Metabolism , Pharmacology , Amino Acid Sequence , Blotting, Western , Cell Line, Tumor , Cell Survival , Dose-Response Relationship, Drug , Exotoxins , Chemistry , Metabolism , Pharmacology , Interleukin-6 , Chemistry , Metabolism , Pharmacology , Molecular Sequence Data , Pseudomonas aeruginosa , Genetics , Metabolism , Recombinant Fusion Proteins , Chemistry , Metabolism , Pharmacology , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
After search at hematopoietic stem cell banks for transplant donors, there may be several donors matched with given standards. To determine the most appropriate donor for a specific patient, the potential donors were analyzed and compared by three methods. The first is cross-reactive group (CREG) antigens, which defined as the public antigens that shared specific serological reaction patterns. The second is residue match theory, which concerned the three residues oriented upward toward the T-cell receptor. The third is comparing HLA three-dimensional structure models. The results of the three methods were not completely accorded in our case. However, some less matched donors could be excluded from the candidates and the range of selection was further reduced. It is concluded that combined application of three methods would contribute in selecting donor for hematopoietic stem cell transplantation in clinics.
Subject(s)
Humans , Cross Reactions , HLA Antigens , Allergy and Immunology , Hematopoietic Stem Cell Transplantation , Histocompatibility Testing , Tissue DonorsABSTRACT
To probe into the expression of alpha subunit for IL-6R at both mRNA and protein levels in human leukemic cells and to discuss its implication in targeted treatment for leukemia with recombinant IL-6-PE40 exotoxin fusion protein mediated by IL-6/IL-6R system, semi-quantitative RT-PCR, sequencing and FCM were used to analyze the gene and protein expression levels of alpha subunit for IL-6R in leukemic cells. Our results showed that not only mRNA but also protein of alpha subunit for IL-6R are highly expressed in the myelogenous leukemic cell lines, the relative expression levels of mRNA were KG-1(1.22) > (1.02) > U266(1.00) > U937(0.99) > HL-60(0.76). Among lymphoblastic leukemic cell lines, Raji expressed a certain amount of alpha subunit mRNA (0.77), whereas its alpha subunit protein was not detected. Expression of alpha subunit mRNA and protein were negative in lymphoblastic leukemic cell lines, HuT28 and CEM, and chronic myelocytic leukemic cell line K562. These results correlate with those of FCM highly. Noteworthily, normal human peripheral blood mononuclear cells expressed hardly protein of IL-6R alpha subunit. So this study provides sufficient experimental evidence that the targeted treatment by recombinant IL-6-PE40 can specifically kill leukemic cells highly expressing IL-6R without toxicity to normal hematopoietic cells.
Subject(s)
Humans , Base Sequence , DNA, Neoplasm , Gene Expression , HL-60 Cells , K562 Cells , Leukemia , Metabolism , Molecular Sequence Data , Protein Subunits , Genetics , RNA, Messenger , Receptors, Interleukin-6 , Genetics , Tumor Cells, Cultured , U937 CellsABSTRACT
The purpose of the research is to provide a new standard for matching of HLA three-dimensional structure, and summarize the major permissible mismatch and immunogenic mismatch antigens. The molecular modeling method was used to create HLA molecular structures by Swiss Model Server, and the comparison of the differences among the alleles was done by SPDV software with the function of iterative magic fit. The results were recorded by relative mean square deviation (RMSD, nm). The differences among alleles were scattered below 0.06 nm for HLA-A and -B molecules, and below 0.03 nm for HLA-DRB1 molecules. On the basis of the statistical analysis, when RMSD is greater than 0.04 nm for -A and -B molecules and 0.02 nm for -DRB1 molecules, the difference is meaningful and can be related with graft versus host disease. When RMSD is lower than 0.02 nm for -A and -B molecules and 0.01 nm for -DRB1 molecules, the difference is decided unmeaningful. From the data, the permissible mismatch and immunogenic mismatch alleles within HLA-A, HLA-B and HLA-DRB1 molecules were summarized. Three-dimensional structure matching is a new area in the transplantation field, much research should be done in the future.