细胞壁缺陷对白喉棒状杆菌Tox基因影响

目的探讨细胞壁缺陷对白喉棒状杆菌毒力基因及其表达的影响。方法以非高渗分离培养法诱导并获得产毒性白喉棒状杆菌稳定L型纯培养物，提取白喉棒状杆菌稳定L型的染色体DNA，用Tox基因特异性引物进行PCR扩增，并进行序列测定和分析。分别采用对流免疫电泳（CIEP）和十二烷基磺酸钠-不连续聚丙烯酰胺凝胶电泳（SDS-PAGE）检测白喉棒状杆菌稳定L型可溶性代谢产物中的白喉毒素蛋白质。结果白喉棒状杆菌在氨苄青霉素作用下可发生细胞壁缺陷而成为L型，该稳定L型的传代培养物可仍然保留同其亲代细菌型一致的Tox基因及其核苷酸序列；但在其可溶性代谢产物中并未检测到白喉毒素蛋白质。结论白喉棒状杆菌稳定L型虽然保留了Tox基因但并不能表达白喉毒素蛋白质，提示细胞壁缺陷可影响Tox基因在宿主菌细胞内的表达。

Effect of cell wall deficiency on Tox gene of Corynebacterium diphtheriae

Objective To probe the affection of the cell wall deficiency to the virulent gene of L-form derived from c.diphtheriae and the expression of the virulent gene.Methods L-forms of c.diphtheriae were induced by ampicillin in the non-high osmotic medium and then the pure cultures of stable L-form were isolated and subcultured in the non-high osmotic medium without any antibiotic.The chromosome DNAs of the pure cultures of the stable L-form were isolated and then the virulent gene was amplified with the Tox gene-specific PCR primers,and were detected by nucleotide sequencing analysis.The diphther-otoxin proteins which isolated from the metabolite of the L-forms of c.diphtheriae were detected by Counter immunoelectrophoresis(CIEP) and SDS Polyacrylamide Gel electro-phoresis(SDS-PAGE).Results The L-forms were induced easily from c.diphtheriae by the ampicillin in non-high osmotic liquid medium.The subcultures of stable l-form derived from c.diphtheriae have the same Tox gene and nucleotide sequence as that of their parental bacteria,But the diphtherotoxin proteins isolated from the metabolite of the L-forms of c.diphtheriae couldn't be detected.Conclusion The stable L-forms derived from c.diphtheriae keep the Tox gene on their chromosome DNA,but they can not express the diphtherotoxin or the toxin proteins.The cell wall deficiency can not result in loss of the Tox gene,but can affect the expression of the Tox gene existed in L-form cells of c.diphtheriae.