| Environmental Estrogen, also known as Endocrine Disrupting Chemicals, is a kind of exogenous chemicals.It can interfere withhormone synthesis, secretion, transport, binding, metabolism, digestion etc., and have a negative influence inmaintaining normal homeostasis, reproduction, growth and behavior to organisms. In addition to acting via genomic process(binding of activated estrogen receptor ER), environmental estrogen can also produce a rapid effect of non-genomicpathways through the membrane estrogen receptor with G protein. A highly expression system was constructed in this study.The recombinant protein was purified in order to produce polyclonal antibody by immunizing rabbits. The transcript and translation level of G protein α subunits underdifferent BPA(1μg/L,10μg/L,50μg/L,100μg/L)and B(a)P(0.5μg/L,10μg/L,50μg/L)exposure in Perinereis aibuhitensis were analyzed, and the results will provide more basis for the toxicity mechanisms about environmental estrogens to P.aibuhitensis.The results are as follows:1. The recombinant expression plasmid Pet-28 a Gα was constructed with ORF of Gα, and E.coli BL21(ED3) was choosen as the host bacteris for recombinant protein expression.Inducing 4h by 1mmol/L IPTG at 37℃was theoptimum conditions. SDS-PAGE showed that recombinant protein exists in inclusion body form. The purified Gα protein was obtained through dissolving the inclusion body by 8M urea and purifying by Ni-NAT column. Gα polyclonal antibody was successfully prepared by immunizing New Zealand rabbit,and good specificity of the Gα polyclonal antibodies was detected by Western blotting. 2. The gene expression of male and female P. aibuhitensisanderexposure to various concentrations of BPA is different. At 14 d, Gα m RNA expression in bodywall of femalewas the most obvious at 100μg/L BPA group, which was6.32 timesto the control group with a significant difference(P<0.01).The most obvious Gα m RNA induction was found in 100μg/L BPA groupin male body wall, andit reached the highest level at 4d, which was 4.65 times to control group. However, Gα m RNA expressions of male bodywall in other concentration groupswere not significant compared with blank control group.The head of male P. aibuhitensiswasmore sensitive than the femal head under diffent BPA concertrations exposure, and the gene expression level were higher than control group in 50μg/L and 100μg/LBPA at 4d and 7d. ELISA results showed that the Gα protein expression in translation level were almostly consistent with the transcriptional level.The translation level of female Gα protein in bodywall rise at 4d、7d and 14 d, and there is a significant time-effect relationship. At 14 d,the translation level of female Gα proteinin bodywall at 100μg/L BPAgroup reached the highest value(P<0.01); the translation level of female Gα proteinin head did not change significantly at 4d and 7d, but at 14 d the expression of 100μg/L BPA group was significantly induced. The translation level of male Gα protein bodywall reached the hightest value at 4d, and the expression at 7d and 14 dreturned to the level which were similar to that in control group; the translation level of male Gα protein in headrise at 4d、7d with a significant difference(P<0.01), and the expression level returned to the level which were similar to that in control group at 14 d.3. The gene expression level of female P. aibuhitensis bodywall under exposure to B(a)P wasnot obviously up-regulated. At 7d, Gα m RNA expression in female head in 50μg/L B(a)P was only 50% compared to control group, and the gene expression did not change significantlyat 4d and 14 d. The Gα m RNA expression of male head were obviously inhibited under B(a)P exposure at 4d which were only 26%,27% and 30% to control grouprespectively; at 7d and 14 d the gene expression returned to normal level which were similar to that in control group and the male head gene expression trendwereconsistent with the male bodywall. ELISA results showed that the Gα protein in female bodywall were significantly higher than the control group(P<0.05) in all concentrations at 7d. At 4d, Gα translation in female head in 5μg/L B(a)P reached the highest level with significant difference(P<0.01). The Gαtranslation in male bodywall was increased with the extension of esposure time and there is a significant time-effect relationship. The translationlevel in male bodywall were significantly lower than that of control group at 4d, and the translationlevel were all higher than the control group at 14 d with a significant difference(P<0.01).The Gα protein translation in male head were consistent with male bodywall and also has a significant time-effect relationship. |
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