Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Expression of a Soluble-modified Green Fluorescent Protein in Eschcrichia Coli and Preparation of Polyclonal Antibody Against It

Author SunChuanBo
Tutor YangZhiJie;WangXingZhi
School Northeast Normal University
Course Zoology
Keywords soluble-modified green fluorescent protein (smGFP) reporter gene chromophore prokaryotic expression
Type Master's thesis
Year 2007
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Soluble-modified green fluorescent protein is a member of GFP variants containing 238 amino acids and its molecular weight is about 30kDa. The protein can catalyze itself into chromophors, and emits stable green fluorescent and fluorescent reaction does not need any substrate or accessory factors, therefore it is easy to be detected. Meanwhile it is expressed independently in cells regardless of the sort they belong to, the location, or their species, and it is no-toxic to receptors and has no influence on gene expression. Recently smGFP has become a standard reporter in many biological systems. In this study, smGFP is highly expressed by prokaryotic expression system,purified in vitro and used as antigen for making its polyclonal antibodies.The smGFP gene from the plasmid pCAMBIA1301-smGFP was inserted into the pET-28a (+) vector between the site of EcorRⅠand HindⅢto create a recombinant plasmid pET-28a(+)-smGFP. Then the recombinant pET-28a (+)-smGFP was confirmed by PCR, restriction, enzyme digestion and DNA sequencing. The recombinant plasmid was transferred into the competent BL21 (DE3) cells and approximately a 30 kDa fusion protein was induced by the addition of isopropyl-β-D -thiogalactoside (IPTG) (1.0 mM) during cultivation. The soluble fusion protein smGFP was purified after expending cultivation by Ni-NTA affinity chromatography under the denaturing condition. The concentration of the fusion protein smGFP was about 700μg /ml determined by Bradford assays. Three 6 to 8 weeks old BALB/c mice were immunized with purified fusion protein smGFP three times using 15μg antigen each time. We have generated the polyclonal antibody. The antibody titration was up to 1:32000 determined by ELISA assays. Western hybridization of antibody and smGFP showed a single band in the nitrocellulose (NC) membrane. These results showed that we have obtained the smGFP protein by in vitro expression system and its polyclonal antibodies.

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