Cloning And Expression Of Staphylococcal Panton-Valentine Leukocidin Gene And Production Of Polyclonal Antibody Against PVL

Objective: To clone and espress lukS-PV and lukF-PVgene from Panton-Valentine leukocidin Positive Staphylococcus aureus, and produce polyclonal antibodies against and LukF-PV of Staphylococcal Panton-Valentine leukocidin. Methods: the primers were designed by the sequence published on PUBMED, including the BamH I and Xho I enzyme cut sites. The gene of lukS-PVand lukF-PV were amplified by polymerase chain reaction (PCR)with the specific primers from the genomic DNA extracted in the reference strain of PVL positive Staphylococcus aureus, and cloned in pGEM-T vector. Then lukS-PV and lukF-PV gene were subcloned in the expression vector pET28a(+). After analyzing by PCR ,restriction enzyme BamHI and XhoI, and sequencing, the recombinant plasmid of lukS-PV- pET28a(+)and lukF-PV pET28a(+)were transformed into Rosetta(DE3)plysS, and induced by IPTG, followed by confirmation of SDS-PAGE and Western blotting. The protein of LukF-PV was purified by Ni2 affinity chromatography and immunized white rabbits. The antiserum obtained was detected by ELISA and Western blotting Results: The length of products of PCR and test of restriction enzyme from the recombinant plasmid of lukS-PV- pET28a(+)and lukF-PV pET28a(+)are 939bp and 978bp respectively . Sequencing results exactly consistent with that in GENEBANK. SDS-PAGE and Western blot analysis confirmed that the E. coli Rosetta(DE3)plysS with recombinant plasmid showed the high level expression of LukS-PV and LukF-PVprotein with the His tag after induction. The tite of antiserum obtained from the white rabbits immunized by the recombinant protein LukF-PV is 10-3. Acrroding the Western blotting the antiserum can be detected by the recombinant LukF-PVL and the PVL positive Staphylococcus aureus. Conclusion: lukS-PV and lukF-PV gene were cloned and the expression vector lukS-PV- pET28(a+)and lukF-PV pET28a(+) were constructed successfully and the protein of LukS-PV and LukF-PV have been expressed in Rosetta(DE3)plysS . The antiserum immunized by the purified rLukF-PV was obtained. The study provides the precondition for further study of PVL imunologic diagnosis.