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Cloning Expression of Selenoprotein W from Chicken and Characterization of Monoclonal Antibodies Against Their Recombinant Proteins

Author: XuMing
Tutor: XuShiWen
School: Northeast Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Chicken Selenoprotein W Monoclonal antibodies The prokaryotic expression Indirect ELISA
CLC: S831
Type: Master's thesis
Year: 2011
Downloads: 45
Quote: 0
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Executive Summary

Selenium is an important trace elements necessary for human and animal life activities. Selenoprotein selenium covalently bound to the protein in a form of selenocysteine. Selenoprotein W (SelW) is a low molecular weight cell selenoproteins. In the different biological SelW the amino acid sequences are highly homologous. Previous studies have shown the SelW were expressed in a variety of tissues and organs, and a higher expression levels in skeletal muscle and brain. The current research mammals and rodents SelW function, SelW having regulating striated muscle metabolism, calcium metabolism and anti-oxidation effect. However, For chicken SelW the function has not been reported. For an in-depth study of the function of the chicken selenoprotein W cDNA under the chicken SelW of primers were designed, containing the complete ORF cDNA sequence was amplified by overlap extension PCR cDNA in Sec codon mutation Sequence analysis results with the reference sequence homology of more than 99%, and mutation products were cloned into the pGEX-6P-1 expression vector, the recombinant expression plasmid pGEX-SelW transformed into Rosetta host strain induced by IPTG, the recombinant fusion protein efficient expression, and are expressed mainly in the form of inclusion bodies, purified recombinant fusion protein by the method of plastic cutting, re-use to the thrombin cut off the GST tag, after the second cut gel purified target protein. Polyclonal antibody by Western blotting showed that the fusion protein with the corresponding the GST-SelW of reaction can produce specific bands, does not react with empty vector after induction of GST-tagged proteins. Purified protein BABL / c mice, cell fusion technology, indirect ELISA screening and cell subclones, two hybridoma, named 1B8 and 4H5. Identified by the monoclonal antibody subclasses kit, were IgM class antibody supernatant titers were 1:6400. Prepared 1B8 and 4H5 ascites monoclonal antibody, ascites titer of 1:12800 and 1:6400. 2 hybridoma chromosome number of 103 ± 5, SP2 / 0 myeloma cells significantly more than 65 to 75. 2 continuous passage 3 months and frozen after recovery did not affect the stability of the antibody-secreting hybridoma cells in vitro. No cross reaction with the GST labels and chicken selenoprotein N. Affinity analysis showed that 2 monoclonal antibody affinity target protein is higher than the recombinant fusion protein and chicken SelW of synthetic peptide and high affinity monoclonal antibody sensitive, stable and fast detection applications, these provide the necessary guarantees. In short, the success of two monoclonal antibodies prepared for the chicken SelW rapid, specific detection detection reagents, and also provide the material basis for the chicken SelW function further research.

Full-text Catalog

The Chinese summary     10-11
English summary     11-13
1. INTRODUCTION     13-20
1.1 Selenium discovery and research     13
1.2 selenoprotein synthesis and regulation     1.3 selenoprotein W 13-14
    the tissue distribution 14-18
1.3.1 SelW,     15 the gene structures
1.3.2 SelW     15-16
1.3.3 SelW amino acid sequence     16
1.3.4 SelW protein structure     16 - 17
1.3.5 food selenium SelW mRNA regulation     17
to 1.3.6 SelW the function     17-18
1.4 hybridoma monoclonal antibody technology     18-19
1.5 the experimental purposes significance     19-20
test methods and materials     20-39
2.1 test reagents and materials     20-23
2.1.1 test reagents     20
2.1.2 Strains and vectors     20
2.1.3 Materials of     20
2.1.4 experimental animals     20
2.1.5 the main reagent preparation     20-23
2.1.6 equipment     23
2.2 experiment     23-31
2.2.1 target gene prokaryotic expression vector construction     23-27
2.2.2 chicken SelW cDNA prokaryotic expression vector construction     27-30 < br /> 2.2.3 recombinant plasmid expression in Escherichia coli induced     30-31
2.3 monoclonal antibody preparation     31-36
2.3.1 antigen emulsion preparation   the   31-32
2.3.2 animal immune     32
2.3.3 indirect ELISA method for detection     32-33
2.3.4 polyclonal antibody specificity identification     33-34
2.3.5 feeder layer cell preparation     34
2.3.6 myeloma cells to prepare     34-35
the lymphocyte preparation 2.3.7 spleen     35
2.3.8 cell fusion     35-36
2.3.9 the positive hybridoma Screening     36 < br /> 2.3.10 positive hybridoma cloning - the limited dilution     36
2.4 monoclonal antibody preparation     36-37
2.4.1 In vitro culture     36
2.4.2 body lure Health ascites method     36-37
2.5 monoclonal antibody characterization     37-39
2.5.1 supernatant and ascites monoclonal antibody titer determination     37
2.5.2 positive hybridoma cells and cryopreservation     37
2.5.3 monoclonal antibody subclasses identified     37
the hybridoma chromosome 2.5.4 the number of identified     37-38
2.5.5 monoclonal antibody secreting antibody stability identified     38
2.5. 6 monoclonal antibody specificity analysis     38
2.5.7 monoclonal antibody affinity and degree of determination     38-39
3 test results and analysis     39 - 52
3.1 the mutant chickens Sel W cDNA Preparation of     39-41
3.1.1 overlap extension PCR     39-40
3.1.2 mutant chicken SelW cDNA gene cloning, characterization and sequencing analysis     40-41
3.2 chicken Sel W cDNA prokaryotic expression vector construct     41-43
3.2.1 purified PCR amplification products and vector pGEX-6P-1 digested   the   identification of 41
3.2.2 recombinant plasmid     41-43
3.3 recombinant protein expression and identification     43-44
3.3.1 recombinant fusion protein induced expression     the general optimization 43
3.3.2 expression conditions     43
3.3.3 recombinant protein soluble analysis     43-44
3.4 GST tag by thrombin and purified target protein     44-45
3.5 purified target protein concentration     45
of 3.6 indirect ELISA reaction conditions The optimized     45-47 the 3.6.1 antigen and serum concentration
determine     45
3.6.2 enzyme labeled antibody concentration to determine     45-46 < br /> 3.6.3 Serum role of time to determine determine nbsp     46
3.6.4 HRP antibody role of time to determine     46
3.6.5 blocking solution to their closing time color;   46-47
3.6.6 Substrate time determine     47
3.7 polyclonal antibody specific identification     47-48
3.8 hybrid establishment of tumor cell lines and partial characterization results   the   48-52
3.8.1 cell fusion rates     48
3.8.2 monoclonal antibody titer determination     48
3.8.3 hybridoma cell lines secrete antibodies stability test results     48-49
3.8.4 Monoclonal antibody subclasses identified     49
3.8. 5 monoclonal antibody chromosome count results     49-50
3.8.6 monoclonal antibody specificity analysis     50
3.8.7 Determination of monoclonal antibody affinity     50-52
4. discussed   nbsp the purpose gene cloning   52-62
4.1;   52
4.2 expression vector select     52-53
4.3 the choice of the host expression bacteria     the conventional optimization nbsp 53
4.4 expression conditions;   53-54
4.5 Expression and purification of the target protein     54
4.6 the indirect ELISA optimization of reaction conditions     54-56
4.7 antigen preparation     the 4.8 experimental animals 56
selection and immune     56-57
4.9 cell fusion     57-58
the 4.10 monoclonal cell lines Screening     58
4.11 hybridoma clones     58-59
4.12 pollution prevention and exclude     59-60
the biological characteristics of the monoclonal antibody 4.13     60-62
4.13.1 monoclonal antibody titer nbsp the;  
60 4.13.2 cells cryopreservation and resuscitation     60
monoclonal antibody 4.13.3 choice of a lot of preparation     60
4.13.4 monoclonal antibody specificity analysis     60-61 the
4.13.5 monoclonal antibody affinity determination   the   61-62
conclusion     62-63
Acknowledgements     63-64
Reference     64-69
study for a master's degree during the academic     69

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Poultry > Chicken
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