Ishag Hassan Z A, Liu Maojun, Yang Ruosong, Xiong Qiyan, Feng Zhixin, Shao Guoqing
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing, 210014 China ; College of Veterinary Sciences, University of Nyala, Nyala, Sudan.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing, 210014 China.
Springerplus. 2016 Jun 17;5(1):769. doi: 10.1186/s40064-016-2358-3. eCollection 2016.
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.
猪鼻支原体是猪的一种机会性致病菌，已被证明可转化细胞培养物，这引起了研究人员的兴趣。维多利亚水母的绿色荧光蛋白（GFP）基因被证明是识别混合群体中转化细胞的重要标记。尚未见使用GFP观察猪鼻支原体（菌株HUB-1）基因转移和表达的报道。我们构建了一个pMD18-O/MHRgfp质粒，其包含p97基因启动子、复制起点、四环素抗性标记以及受p97基因启动子控制的GFP基因。该质粒以约4×10⁻³cfu/μg质粒DNA的频率转化猪鼻支原体，并且可通过对转化后的支原体总DNA进行GFP基因的PCR扩增来检测。对在含四环素的KM2琼脂上生长的单个克隆进行分析，显示出绿色荧光。总之，本报告表明GFP可用于监测猪鼻支原体中的瞬时基因转移和表达，最终最大限度地减少基因转移和表达的筛选程序。
