Prevention Of Gastrointestinal Lead Poisoning Using Recombinant Lactococcus Lactis Expressing Human Metallothionein-i Fusion Protein

Objective: Construct human metallothionein-I fusion protein GST-SUMO-MT in plasmid p MG36 e, and transformed it into food-grade L.lactis strain MG1363 by electroporation, to study the preventive effect of chronic lead poisoning.Methods: The deoxyribonucleic acid(DNA) fragments encoding GST-SUMO-MT fusion proteins were inserted into p MG36 e to obtain recombinant L.lactis expression plasmids p GSMT. The stability of recombinant plasmids in the recombinant strains was tested by subculture in M17-Bouillon medium with 0.5% glucose(GM17) in the presence or absence of erythromycin. Evaluating the lead-binding capacity of recombinant strain by Atomic Absorption Spectroscopy. To explore the optimal condition in in a 10 L jar fermenter. Three dose of recombinant L.lactis p GSMT/MG1363 was orally administered to pubertal male Sprague-Dawley rats with chronic lead poisoning, in contrast to those administered DMSA and p GS/MG1363. Biochemical parameters in serum and lead accumulation in tissues were detected to evaluate the preventive effect of lead poisoning. Acute toxicity test and skin irritation test were performed to assess the safety of recombinant L.lactis p GSMT/MG1363.Results: Objective protein GST-SUMO-MT was expressed in food-grade L.lactis strain MG1363. In the continuous culture experiment with or without erythromycin, approximately 69.5 ± 3.5% erythromycin-free-cultured clones survived on the GM17 medium containing erythromycin and all erythromycin-cultured clones survived, showing that the recombinant p GSMT plasmid was stable in L.lactis. The atomic absorption spectrometric analysis data suggest that total lead-binding capacity of p GSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass when cultured for 20 h, which p GS/MG1363 and MG1363 was 2.631 ± 0.28 mg/g, 2.181±0.070 mg/g, respectively. Under optimal condition(30 ℃, p H6.0, 100 rpm, 1.0 vvm), 30-L fermentation can harvest 3.175± 0.83 g/L dry mass(6×1011 Colony-Forming Units),which reaches production demands. Continuously oral administered of three dose level recombinant strains decreased lead absorption by 5%, 46.22% and 59.22%, respectively, compared with the model group. The atomic absorption spectrometric analysis data suggest that three dose level recombinant strains provided significant prevention of lead accumulation in kidney liver and brain in a dose-dependent manner. The effcet of high-dose p GSMT/MG1363 didn’t show significant difference between DMSA group(p >0.05). After lead exposure for 6 weeks, the average alanine aminotransferase(ALT), aspartate aminotransferase(AST) and total bile acid(TBA) levels of the model group were 202.8 ± 15.2 IU/L, 395.4 ± 33.1 IU/L, and 19.5 ± 1.7 μmol/L, respectively, which were significantly higher than those of the control(p< 0.001). Although the accumulation of lead in the kidneyis highest than that of other soft tissues, there were no significant changes in the creatinine and urea levels between the control group and lead-treated rats with or without L. lactis. By contrast, lead elevated the serum urea acid 3.8 times higher than that of the control. Exposure to lead also induced elevated creatine kinase(CK) concentrations in the serum. Treatment with recombinant L. lactis for 6 weeks did not result in significant differences for the concentrations of calcium, magnesium, sodium, chlorine or potassium ion. The I125-based radioimmunoassay(RIA) results demonstrated that lead significantly suppressed testosterone synthesis and secretion. The average testosterone level of the lead-treated rats decreased by 84.5% compared with that of the control rats(p< 0.001). However, high-dose p GSMT/MG1363-treated and DMSA-treated rats maintained the normal serum concentration of testosterone. Acute toxicity test results suggest that recombinant strains were non-toxic and skin irritation test average score was 0.0238, which shows nonirritant.Conclusions: The fusion protein GST-SUMO-MT expressed in Lactococcus lactis strain can prevent tissues like blood, kidney and kidney from the damage induced by lead exposure in male rats and it’s safe. The recombinant Lactococcus lactis strain p GSMT/MG1363 can effectively inhibit the absorption of poisoning heavy mental ions via gastrointestinal tract.