| As we know, heavy metal pollution in water become more and more serious along with the rapid development of agricultural, industrial and military operations. Heavy metals are, unlike other pollutants, difficult to be removed from the environment and cannot be chemically or biologically degraded. Metallothioneins (MTs), found in a large number of phylogenetically diverse organisms, are a super-family of small, cysteine-rich, metal-binding proteins that can bind a range of metals. They play a major role in heavy metal detoxification, essential metal homeostasis and antioxidation. The freshwater crab Sinopotamon henanense (S. henanense) is commonly found in southeast Shanxi Province, an area that has endured the most serious environmental pollution. It lives in the sediment and faces heavy metals directly via its integument and via food. In our previous report, S. henanense showed a strong capability of accumulating heavy metals. A novel MT from S. henanense has been purified and its full length gene has been isolated from the cDNA library of S. henanense.In order to further elucidate the mechanism of how the crab MT is effective in heavy metal detoxification and the metal-binding abilities and develope an ELISA for it, the research of this thesis is divided into the flowing parts:Chapter One, to produce of the recombinant MT of S. henanense, the gene for the crab MT was over-expressed as fusions with SUMO in Escherichia coli (E. coli) and purified by affinity chromatography. Chemical and functional characteristics of the purified MT were investigated. Subsequently, the BALB/c mice were immunized with the recombinant MT and the resulted antiserum was analyzed by ELISA, Western blotting and immunohistochemical stainting. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal binding capacity of MT. Results showed the high purity MT was obtained in the soluble fraction by SUMO fusion protein system and exhibited intact metal binding ability and hydroxyl radical scavenging ability. The antiserum of high titer against MT of S. henanense was prepared in mice and was proved to have a good specificity by Western bloting and immunohistochemical stainting. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn2+, Cu2+and Cd2+in these strains followed the decreasing order of SUMO-MTtl>SUMO-MTt2> SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. The present study could help to elucidate the mechanism of how the crab MT is effective in heavy metal detoxification and provide a scientific basis for development of an ELISA for determination of MT.Chapter Two, the SUMO-MT fusion protein was over-expressed inside E. coli and purified by affinity chromatography following a method previously described by our group. The recombinant SUMO-MT and MT from digestion of SUMO-MT were subsequently used as immunogen and detecting antigen to screen antibodies produced by hybridoma cells against the MT of S. henanense. The titers of mAbs were measured by indirect ELISA and the specificities of the mAbs were evaluated by Western blot and Dot-ELISA assays. The results showed that two hybridoma cell lines were screened, designated mAb-MT2 and mAb-MT3, which secrete stably mAbs against MT. Their immunoglobulin subclasses were IgG1. The ELISA titers of the ascites fluid were 1:5×105 and 1:1×106, respectively. Westerm blot analysis confirmed that the two mAbs both reacted with SUMO-MT and digested MT with good sensitivity. The Dot-ELISA results demonstrated that the two mAbs only with MT and not SUMO and His tag. The mAbs against MT of freshwater crab (S. henanense) with high specificity were successfully prepared, which has laid the foundation for further development of immunological detection and determination of MT.Chapter Three, there are more than one MT isoforms in most vertebrates and invertebrates and different MT isoforms play different accurate roles in the physiological processes, according to their metal-binding characteristics. S. henanense is a decapod crustacean widely distributed in the freshwater environments in northern China, in which only one MT isoform has been reported so far. In order to investigate the metal-binding characterization of MT, in this study, we produced recombinant non-fusion MT by a novel secreted expression method using phoA vector in E. coli. The non-fusion MT proteins were then purified using monoclonal antibody (mAb) that we prepared previously, and had a high affinity for both recombinant and endogenous MT. The functional characteristics of the non-fusion MT were analyzed, including the hydroxyl radical scavenging ability and the metal-binding abilities. We found that the non-fusion MT had hydroxyl radical scavenging ability, and had ability to bind Zn, Cu and Cd ions with different metal-binding affinity in E. coli in the order of Zn>Cu>Cd. Moreover, after recombinant MT proteins were reconstituted with different metals, the corresponding metal complexes were confirmed by UV absorption and the abilities of its further were analyzed in vitro. The results showed the metal-binding capacity of MT protein in vitro was not in agreement with in vivo. We found that MT was an unspecific MT and had consistent affinities toward Zn, Cu and Cd in vitro. Our results suggest that the MT protein can bind various metal to form complex of one MT to six bivalent metal by stoichiometries of two M(Ⅱ)3Cys9. These present studies could help further elucidate the accurate functions of MT.Chapter Four, to utilize the MT protein as a useful biomarker for environmental contamination, we developed an enzyme-linked immunosorbent assay (ELISA) in the freshwater crab (S. henanense). New zealand rabbit was immunized with the purified SUMO-MT to prepare polyclonal antisera and then the antisera was purified by rProteinA colum. The titer of the polyclonal antibody was measured by indirect ELISA and the specificity was evaluated by Western blot. Then a sandwich ELISA for determination of MT in the freshwater crab S. henanense was developed using the non-fusion MT as standard protein, in which the monoclonal and ployclonal antibody previously produced were chosen as a capture antibody and detect antibody, respectively. The results showed the standard linear equation, y= 0.0035x+0.0068 (R2=0.9981, x and y represent MT concentration and OD490 value, respectively), was established for the determination of MT concentration with the valid rang of 40-1200 ng/mL. The susceptibility of the ELISA was about 20.0 ng/mL of MT concentration. The results demonstrated the mean coefficients of variation of intra-assay and inter-assay were 4.22% and 4.09%, respectively. In conclusion, the developed ELISA of this study is precise, stable and repeatable. |
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