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为研究淋病奈瑟菌CRISPR系统中Cas蛋白的结构与功能,以WHO-A株基因组DNA为模板,利用PCR技术克隆出CT和TM 2个Cas基因,在大肠埃希菌中进行原核表达。分别用pCold-TF表达的CT和TM重组蛋白免疫小鼠制备Cas抗体;以pGEX-6p-1表达的CT和TM重组蛋白为检测抗原测定免疫血清中特异性抗体的效价。结果表明:获得了与预期分子量大小一致的重组抗原,间接ELISA检测表明,小鼠免疫血清中CT和TM抗体的效价分别为1∶25 600、1∶12 800。结果说明为细胞外探索淋病奈瑟菌中Cas蛋白的作用机制和细胞内检测相关蛋白的含量提供了试验材料。
In order to study the structure and function of Cas protein in Neisseria gonorrhoeae CRISPR system, two Cas genes of CT and TM were cloned by PCR using the WHO-A strain genomic DNA as a template and prokaryotic expressed in Escherichia coli. Cas antibody was prepared by immunizing mice with CT and TM recombinant proteins expressed by pCold-TF, respectively. CT and TM recombinant proteins expressed by pGEX-6p-1 were used as detection antigen to determine the titer of specific antibodies in immune serum. The results showed that the recombinant antigen with the expected molecular weight was obtained. Indirect ELISA showed that the titers of CT and TM antibodies in mouse serum were 1:25 600 and 1:12 800, respectively. The results provide experimental materials for exploring the mechanism of extracellular activity of Cas protein in Neisseria gonorrhoeae and the content of intracellular detection related proteins.