| ObjectivesEscherichia coli prokaryotic expression system was utilized to express the recombinant protein aroA-CC-M, the purity of which is more than95%. After determination of Equivalence of the recombinant protein aroA-CC-M expressed in E.coli and theory protein, the edible safety of the target protein was assessed in two areas of toxicity and allergenicity. Through nutrients’ composition analysis and animal feeding experiment, the impact of GM corn on the growth and development, subchronic toxicity and state of nutrition and health of animals were observed to assess the edible safety of GM corn Bt-799with CrylAc-M gene.Methods1. Expression, purification and equivalence analysis of recombinant glyphosate-resistant protein aroA-CC-M1.1Expression and purification of recombinant protein aro A-CC-MThe recombinant vectors with aroA-CC-M gene were used to transform and expressed in E.coli Rosetta(DE3) under induction of IPTG. After concentration and purification of recombinant protein aroA-CC-M by affinity chromatography, SDS-PAGE was used to determine the purity.1.2Equivalence analysis of recombinant protein aro A-CC-MEquivalence analysis of recombinant protein aroA-CC-M and theory protein was determined in respect of molecular weight assay, N-terminal amino acid sequence analysis, immunogenicity and antigenicity.2. Safety assessment of recombinant glyphosate-resistant protein aro A-CC-M2.1Toxicity study2.1.1Bioinformatics analysisBioinformatics was conducted to determine the homology of recombinant protein aroA-CC-M and known toxic proteins or anti nutrients. 2.1.228-day feeding experimentRecombinant protein aroA-CC-M and the control protein were expressed in Escherichia coli prokaryotic expression system. After expression,the protein was concentrated to the yield0.25mg/ml,0.5mg/ml, l.Omg/ml as low-dose group, middle-dose group and high-dose group. The protein expressed by the recombinant vectors without gene is the control group.80weanling SD rats were randomly divided into four groups based on weight, Three groups of rats were administered recombinant protein aroA-CC-M by gavage at doses of2.5mg/kg,5mg/kg and10mg/kg bw/d for28d. During the experiment, food intake and weight were measured once a week.1h before sacrifice, rats were administered protein by gavage once. At the end of the experiment, blood of each rat was taken to determine the blood routines, blood biochemistry, classification of lymphocyte immunophenotypic and content of protein aroA-CC-M in blood. All rats were sacrificed at the end of the experiment, routine pathological examination and organ coefficient for main viscera were carried out.2.2Allergenicity study2.2.1Bioinformatics analysisBioinformatics was conducted to determine the homology of recombinant protein aroA-CC-M and known allergenic proteins.2.2.2Digestion testDigestion test of recombinant protein aroA-CC-M in simulate gastric and intestinal fluid was performed to know the stability against digestion.3. Safety assessment of genetically modified corn Bt-799with Cry1Ac-M gene3.1Nutrients’analysisProtein, fat, fiber, vitamin, minerals and amino acid in genetically modified corn Bt-799with Cry1Ac-M gene were determined and compared with relevant data in literature.3.2Anti nutrients’analysisTo analyze the anti-nutrtion of GM corn Bt-799, bioinformatics was conducted to determine the homology of protein Cry1Ac-M in GM corn Bt-799and known anti nutrients in corn, such as Phytic acid, Insulin degrading enzyme inhibitors, Chymotrypsin inhibitor etc.3.3Subchronic toxicity assessment(90-day feeding experiment)140weanling SD rats were randomly divided into seven groups based on weight: normal control group, three GM corn groups (each containing12.5%,25%,50%GM corn) and three parental corn groups(each containing12.5%,25%,50%parental corn). During the experiment, food intake and weight were measured once a week. In the middle of and at the end of the experiment, blood routines and blood biochemistry were measured. At the end of the experiment,urine and blood of each rat were taken to determine the urine routine, coagulation index (PT, APTT), classification of lymphocyte immunophenotypic inblood, hormone levels. All rats were sacrificed at the end of the experiment, routine pathological examination and organ coefficient for main viscera were carried out.Results1. Expression, purification and equivalence analysis of recombinant glyphosate-resistant protein aroA-CC-M1.1Expression and purification of recombinant protein aroA-CC-MRecombinant protein aroA-CC-M was obtained by Escherichia coli prokaryotic expression system. After purification and concentration, a lot of soluble recombinant protein aroA-CC-M was obtained. The purity is more than95%, and the yield is about15.6mg/L bacteria fluid。1.2Equivalence analysis of recombinant protein aroA-CC-MMolecular weight, N-terminal amino acid sequence of recombinant protein aroA-CC-M and theory protein were all parallel. The immunogenicity and antigenicity of the recombinant protein aroA-CC-M is the same as expected.2. Safety assessment of recombinant protein aroA-CC-M2.1Toxicity study2.1.1Bioinformatics analysisThe proteins which were homologous to recombinant protein aroA-CC-M were all anti-herbicides proteins. There were no obvious records that could show any harmful effect of these proteins.2.1.228-day feeding experimentThe results of28-days feeding experiment showed that there were no statistical difference between recombinant protein groups and control group in terms of diet intake and bodyweight. Although some indexes of blood routines, blood chemistry, organ coefficient were significant different from control group, majority of the indexed were within normal range reported by other researchers. No obvious abnormality was found in pathological examination.2.2Allergenicity study2.2.1Bioinformatics analysisThe results of Bioinformatics analysis showed that there was no homology of recombinant protein aroA-CC-M and known allergenic proteins.2.2.2Digestion testThe results of digestion test showed that recombinant protein aroA-CC-M was easily digested in gastric fluid, but stayed stable for60min in intestinal fluid.3. Safety assessment of genetically modified corn Bt-799with CrylAc-M gene3.1Nutrients’analysisThere was no statistical difference between genetically modified corn Bt-799with Cry1Ac-M gene and relevant data in literature in respect of protein, fat, fiber, vitamin, minerals and amino acid etc.3.2Anti nutrient’analysisThere were no homology of protein Cry1Ac-M in GM corn with Cry1Ac-M gene and known anti nutrients in corn, such as phytic acid, insulin degrading enzyme inhibitors, chymotrypsin inhibitor etc.3.3Subchronic toxicity assessment(90-day feeding experiment)The results of subchronic toxicity assessment showed that there were no statistical difference between GM corn groups and parental corn groups or normal control group in terms of diet intake and bodyweight. Although some indexes of blood routines, blood chemistry, organ coefficient were significant different from parental corn groups or normal control group, majority of the indexed were within normal range reported by other researchers. No obvious abnormality was found in pathological examination.Conclusion1High purity (>95%) recombinant protein aroA-CC-M was expressed in Escherichia coli prokaryotic expression system. After expression, the target protein was purified with Ni-NTA. The condition of expression and purification was studied and optimized. The yield of the recombinant protein aroA-CC-M reached15.6mg/ml bacteria liquid, and the target protein is equivalent to the theory protein.2There was no homology of recombinant protein aroA-CC-M and known toxic proteins or anti nutrients. The results of28-day feeding experiment showed that there was no obvious toxicity of recombinant protein aroA-CC-M to rats. Recombinant protein aroA-CC-M was easily digested in gastric fluid, but stayed stable for60min in intestinal fluid.3There were no statistical difference between genetically modified corn Bt-799with CrylAc-M gene and relevant data in literature in nutrients composition. There was no homology of protein Cry1Ac-M in GM corn with Cry1Ac-M gene and known anti nutrients in corn. So no obvious influence on the nutrition of genetically modified corn Bt-799could be produced by Cry1Ac-M gene. The results of90-day feeding experiment showed that there was no obvious toxicity of genetically modified corn Bt-799to rats, so there is high food safety of genetically modified corn Bt-799.
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