| According to GB/T 4789.30-2008,38 Listeria monocytogenes isolates were identified from meat, eggs, milk and seafood purchased from supermarkets. The 38 isolates can be divided into 3 parts by multiple-PCR serotyping analysis, among them 34 isolates belong to 1/2a & 3a,3 isolates belong to 1/2c & 3c, and 1 isolate belongs to 1/2b,3b & 7. Drug susceptibility tests showed that most of 38 isolates were sensitive to 30 antibiotics by an agar plate disc diffusion mothod. With mitomycin C induction, a lysogenic phage was induced from one of the 16 isolates harboring prophages at tRNAArg gene. Results of hemolytic activity test showed that 12 isolates had highly hemolytic activities.After the nucleotide sequence of prfA regulon was sequenced from the isolate HA 1069 which has highly hemolytic activity, susceptibility to antibiotics often used in lab and no prophages. The homologous recombination arm for actA/plcB deletion was amplified by SOE-PCR, and cloned into E. coli-L. monocytogenes shuttle plasmid pKSV7, resulting the homologous recombination plasmid pK1013, the vector pK1013 was transformed into HA1069 competent cells by electroporation. the chloramphenicol resistant clones were propagated on solid BHI media with or without antibiotics at 28℃or 41℃for several passages, at last, a chloramphenicol sensitive clone was obtained, and PCR analysis confirmed this clone was an actA/plcB double null mutant.DNA fragment encoding chimeric antigen 856A2 of Mycobacterium tuberculosis joined with the promoter of hly was cloned into pKSV7 in the proper orientation, and then transformed into actA/plcB mutant. The positive recombinants were confirmed by PCR. Western blot result showed that the target protein 856A2 was detected in E.coli BL21 carrying pet28a-856A2 as positive control, but not in actA/plcB mutant harboring the plasmid pKh856A2, this implied that the protein 856A2 secreted into the supernatant of actA/plcB mutant culture is much less, so not enough for Western blot detection. |
