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2022, Vol. 26 ›› Issue (2): 171-175Identification and expression of exogenous target genes in transgenic tomatoGuan Weiwei1, 2, 3, Gu Yu2, Guan Xiaoyan2, 3, Wu Jiayuan2, 3, Bai Guohui3, Tian Yuan2, 3, Liu Jianguo2, 3
Abstract: BACKGROUND: Edible vaccines from transgenic plants is to integrate exogenous genes into the plant genome with plants as carriers, further activating animal or human immune system to obtain specific immunity. However, the continuous low expression of exogenous genes has been unable to achieve satisfactory immune effects. OBJECTIVE: To detect the foreign fused gene pacA-ctxB gene and interest protein expression level in the transgenic tomato by molecular biological technique, providing a research basis for further observation of the anti-caries effect of edible caries vaccines. METHODS: After extracting the total DNA of transgenic tomato leaves, the exogenous fused gene pacA-ctxB was detected by PCR. There were three groups in the experiment: a positive control group (plasmid p2355-EPC10), a blank control group (an ordinary tomato plant, Hongkang 219), and a transgenic group (nine transgenic tomato plants). Total proteins were extracted and quantitatively tested with BCA kit and expression of PAcA/CTB in the transgenic tomato was analyzed by western blot and ELISA. There were two groups for detection of target proteins: a transgenic group (five transgenic tomato plants expressing exogenous chimeric genes (positive PCR test) and a blank control group (one ordinary tomato plant). RESULTS AND CONCLUSION: PCR amplification results showed that there were 5 of the 9 strains of transgenic tomatoes in which the specific amplification bands were about 1.7 kb, accounting for 55% of the total detected transgenic plants. The total protein in the transgenic tomato was 3.15 g/L. Western blot results showed high density bands at 5.8 kD for PCR-positive transgenic tomato protein samples, and no specific bands were found in non-transgenic tomato protein samples. ELISA results showed that the expression of target proteins was about 4.12 mg/L, accounting for 0.13% of the total soluble protein in the transgenic tomato. To conclude, the exogenous fusion gene pacA-ctxB can be expressed in tomato plants and produce target proteins. Key words:anti-caries vaccine, transgenic tomato, decayed tooth |