通过替代失活的方法分析核酸片段在肺炎链球菌毒力中的作用

目的鉴定基因spd—ABC、spd-1672、sp-1673和长间隔序列spd—J在肺炎链球菌（Streptococcus pneumoniae，S．pn）毒力中的作用。方法采用长臂同源聚合酶链反应（LFH—PCR）的方法分别构建这4个序列的红霉素抗性基因（erm）替代缺失突变体，通过PCR和测序鉴定是否构建成功；通过绘制生长曲线观察基因对细菌生长繁殖的影响，通过小鼠毒力实验观察基因对细菌致病性的影响。结果所构建缺陷菌目的基因由eFm基因完全替代；spd—ABC、spd-1673和长间隔序列spd—J3个片段缺陷菌的生长趋势与野生菌没有明显差异，生长大约5hA值均可达到峰值，而spd-1672缺陷菌出现明显的延迟现象，生长8h后其4值才达到最高值；野生菌与缺失spd—ABC、spd-1673和长间隔序列spd—J的缺陷菌感染小鼠各组半数死亡时间分别为19、22、24和24h，差异无统计学意义，而spd-1672缺陷菌感染小鼠的半数死亡时间在75h左右，显著长于野生菌感染小鼠组（P〈0．05）。结论在构建的4个单个基因缺失突变体中，spd-1672缺陷后．S．pn生长明显延迟，毒力显著下降，提示spd-1672是s-Pn的一个新的毒力因子。

Analysis of the Role of DNA Fragments in Streptococcus Pneumoniae Virulence Through the Methods of Alternative Inactivation

Objective To determine the effects of the genes spd-ABC, spd-1672, spd-1673 and the long interval sequence spd-J on S. pn virulence. Methods Four erythromycin resistance gene （erm） substitution mutants were constructed by long arm of homology polymerase chain reac- tion （LFH-PCR） method and then identified by PCR and sequencing. The effects of the mutant genes on S. pn growth were investigated by growth curve assay. The role of the mutants in the patho- genicity of S. pn was evaluated by Virulence experiment. Results PCR and sequencing results showed that all defected mutants were successfully constructed and completely replaced by erm genes. There was no significant difference in growth curves between spd-ABC, spd-1673, spd-J mutants and wild strain, overall appearing growth peak （OD value） till 5 hours. However, the growth peak of spd-1672 mutant was significantly delayed 8 hours late. Virulence experiments showed that the medium lethal time of wild strain and mutants of spd-ABC, spd-1673, spd-J groups were 19h, 22h, 24h, 24h respectively, with no statistical difference. On the other hand, the medium lethal time of spd-1672 mutant group was about 75 hours, significantly longer than that of wiht strain group （ P 〈0. 05） . Conclusion Of the four mutants with single gene deletion, S. pn showed obvious delay in growth and significant decrease in virulence when spd-1672 was depleted, suggesting that spd-1672 is novel virulence factor.