Wei sheng yan jiu = Journal of hygiene research · 2005
[Study on the food safety of the marker gene expressed protein HPT].
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Yang Li-Chen, Li Ying-Hua, Yang Xiao-Guang, Piao Jian-Hua
Аннотация
OBJECTIVE: This article studied on the food safety of HPT protein (Hygromycin B Phosphotransferase), a kind of plant selectable marker gene expression product, which mainly included the quantity and quality detection of HPT in the SCK transgenic rice plants, the possible dietary intakes and its stability in the simulative digestive system.
METHODS: HPT cDNA fragment was inserted into the prokaryotic expressing vector pBV222 for the expression of 6His-HPT fusion protein. The inclusion bodies were denatured by 8 mol/L urea and purifiee with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT were used to immunize rabbit and BALB/C mice for the preparation of the polyclonal and monoclonal antibodies, and the double-Ab sandwich ELISA to detect HPT was established then. Concerning the sensitivity assessment of HPT protein, the amino acid sequences of HPT was compared with the other allergens and the simulative digestive experiment in vitro was carried out too.
RESULTS: 6His-HPT protein was obtained with the purity up to 95%. The specificity of antisera and ascites was detected by Western blot, which showed specific binding reaction between the antibodies to the purified 6His-HPT proteins and their expressed product. The double-Ab sandwich ELISA system was established successfully with the sensitivity of 30 ng/ml. The HPT expression level in the leaves of transgenic rice was proved in the range of 80 - 150 ng/ml, yet we cannot detect HPT protein in the seeds of the same rice. There showed no comparability in the amino acid sequences between HPT and other allergens, and HPT protein was digested quickly in the simulated stomach and intestines liquids.
CONCLUSION: All the results showed that the expression level of HPT in the SCK transgenic rice plants is very low and they are not stable in the simulative digestive system. So we inferred that under the current detection level, HPT protein might not be able to induce any safety problem. Yet further study on the acute toxicity and sensitivity experiments by the animals need to be carried out, and the results will be reported in the near future.
METHODS: HPT cDNA fragment was inserted into the prokaryotic expressing vector pBV222 for the expression of 6His-HPT fusion protein. The inclusion bodies were denatured by 8 mol/L urea and purifiee with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT were used to immunize rabbit and BALB/C mice for the preparation of the polyclonal and monoclonal antibodies, and the double-Ab sandwich ELISA to detect HPT was established then. Concerning the sensitivity assessment of HPT protein, the amino acid sequences of HPT was compared with the other allergens and the simulative digestive experiment in vitro was carried out too.
RESULTS: 6His-HPT protein was obtained with the purity up to 95%. The specificity of antisera and ascites was detected by Western blot, which showed specific binding reaction between the antibodies to the purified 6His-HPT proteins and their expressed product. The double-Ab sandwich ELISA system was established successfully with the sensitivity of 30 ng/ml. The HPT expression level in the leaves of transgenic rice was proved in the range of 80 - 150 ng/ml, yet we cannot detect HPT protein in the seeds of the same rice. There showed no comparability in the amino acid sequences between HPT and other allergens, and HPT protein was digested quickly in the simulated stomach and intestines liquids.
CONCLUSION: All the results showed that the expression level of HPT in the SCK transgenic rice plants is very low and they are not stable in the simulative digestive system. So we inferred that under the current detection level, HPT protein might not be able to induce any safety problem. Yet further study on the acute toxicity and sensitivity experiments by the animals need to be carried out, and the results will be reported in the near future.