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目的:在获得含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄原代种子的基础上,应用分子生物学技术检测外源基因在转基因植株中的表达,测定目的蛋白的含量。方法:PCR筛选含编码变异链球菌表面蛋白PAcP与霍乱毒素B亚单位融合基因的转基因番茄植株;提取番茄果实总蛋白,用BCA试剂盒测定番茄果实总蛋白含量;通过Western印迹检测外源蛋白的表达情况,并用ELISA法对外源目的蛋白含量进行测定。结果:PCR扩增分析可见1.6 kb特异性扩增条带,出现特异性条带的植株占总检测植株的55.6%;转基因番茄总蛋白含量为3.93 mg/mL,Western印迹结果显示,在PVDF膜上,约60 kD处出现特异性条带;ELISA测得表达的目的蛋白占番茄可溶性总蛋白的0.18%。结论:含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄子代植株能有效表达外源蛋白。
OBJECTIVE: To detect the expression of foreign genes in transgenic plants by molecular biology techniques after obtaining the primary transgenic tomato plants containing the fusion gene of Streptococcus mutans surface protein PAcP and cholera toxin B subunit. content. Methods: PCR was used to screen transgenic tomato plants containing fusion gene of PAcP and cholera toxin B subunits of Streptococcus mutans; total protein of tomato fruits was extracted; total protein content of tomato fruits was determined by BCA kit; The expression of the target protein was determined by ELISA. Results: The 1.6 kb specific amplified bands were found by PCR amplification, 55.6% of the total plants were detected by specific bands, and the total protein content of transgenic tomato was 3.93 mg / mL. Western blotting results showed that in the PVDF membrane , A specific band appeared at about 60 kD. The expressed protein of interest expressed 0.18% of total soluble tomato protein. CONCLUSION: Transgenic tomato progeny plants containing the fusion gene encoding Streptococcus mutans surface protein PAcP and cholera toxin B subunits are efficient in expressing foreign proteins.