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 purecultures 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 Polyacry lamide Gel electro-phoresis(SDS-PAGE).

  Results   The Lforms were induced easily from c.diphtheriae by the ampicillin in non-high osmotic liquid medium.The subcultures of stablel-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.