MOLECULAR CLONING OF PNEUMOLYSIN GENE FROM S. PNEUMONIAE TYPE 19F

第19群肺炎球菌溶血素基因的分离和分子克隆 染色体DNA，使用32 P标记的克隆肺炎球菌溶血素 以1型肺炎球菌溶血素基因为探针（发现2型肺炎球菌溶血素基因 显示与1型肺炎球菌溶血素基因相似核苷酸序列，具有6- 1,530个核苷酸之间的位置差异）。 第19组染色体 分离DNA样品并用各种限制性内切酶处理。 将DNA样品印迹到硝酸纤维素滤膜上，并与一个杂交探针杂交。 32 P标记探针。 19 F、19 A、19 B和19 C型染色体DNA在染色体上显示2条带， 放射自显影照片，用EcoRI、Hind III和 EcoRV酶。 相比之下，除了19 A型，这些DNA样本 当用ClaI处理时，仅显示1条带，19 F和19 C为5 kb， KB为19 B。 19 A型DNA显示2条带，分别为2.2和3.5 kb。 因此，第19组肺炎球菌产生肺炎球菌溶血素分子， 显示出相同的溶解红细胞的生物活性，但可能具有 肺炎球菌溶血素基因中的突变。 通过将ClaI产生的19 F DNA克隆到 来源于pUC 19的噬菌粒载体的ClaI位点。 一个5 kb的片段 分离19 F DNA，连接到载体的ClaI位点， 克隆到E. coli DH5a。 转化的DH 5a细胞含19 F5， kbDNA表达的肺炎球菌溶血素活性较高。 类似的研究 继续连接来自ClaI处理的染色体DNA的DNA片段 将其它组19的基因克隆到载体上，并克隆到E. coli细胞。 限制性内切酶位点和肺炎球菌溶血素的遗传特性 基因DNA片段，分离自ClaI处理的染色体DNA样品 第19章将进一步研究

Isolation and molecular cloning of the pneumolysin gene from group 19 chromosomal DNA were performed, using 32P-labeled cloned pneumolysin gene from type 1 strain as a probe (type 2 pneumolysin gene was found to show similar nucleotide sequence as type 1 pneumolysin gene, having 6- location difference among 1,530 nucleotides). The group 19 chromosomal DNA samples were isolated and treated with various restriction enzymes. DNA samples were blotted to nitrocellulose filters and hybridized with a 32P-labeled probe. Type 19F, 19A, 19B and 19C chromosomal DNA exhibited 2 bands in the autoradiograph pictures, following treatment with EcoRI, Hind III, and EcoRV enzymes. In contrast, except for type 19A, these DNA samples showed only 1 band, when treated with ClaI; 5 kb for 19F and 19C, and 9 kb for 19B. Type 19A DNA showed 2 bands, which were 2.2 and 3.5 kb. Thus, group 19 pneumococci have produced pneumolysin molecules, which show the same biological activity to lyse erythrocytes, but may have differeces in their pneumolysin genes. A gene library of ClaI generated 19F DNA was constructed by cloning into the ClaI site of a phagemid vector derived from pUC19. A 5 kb fragment of 19F DNA was isolated, ligated into a ClaI site of the vector, and cloned into E. coli DH5a. The transformed DH5a cells containing 19F 5 kb DNA expressed high pneumolysin activity. Similar studies were continued to ligate the DNA fragment from ClaI-treated chromosomal DNA of other group 19 to the vector and clone into E. coli cells. Genetic characterization of restriction enzyme sites and pneumolysin gene DNA fragments, isolated from ClaI-treated chromosomal DNA samples of group 19 will be studied further.