Progress in research of the DrrAB efflux system of Streptomyces peucetius / 中国人兽共患病学报

The bacterium Streptomyces peucetius produces doxorubicin and daunorubicin,and the two widely used anticancer antibiotics.Two open reading frames,drrA and drrB,were proposed to encode for an ABC (ATP-binding cassette) type of permease that carries out export of the antibiotic ABC (ATP binding cassette)-type transporter for the exportation of these two antibiotics.In this paper,the structure,expression and interaction of DrrA and DrrB protein and the research progress of the assembly and multi drug efflux function of DrrAB are briefly reviewed.

Cloning and expression of Staphylococcus aureus surface protein Isdb and its immune experiment in mice / 生物工程学报

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface Isdb, we amplified Isdb gene from S. aureus Wood46 strain. The isdb gene was subsequently inserted into pET32a(+) vector and the recombinant plasmid was transformed into E. coli strain BL21. The recombinant Isdb was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level was measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus strains Wood46 and HLJ23-1. These results showed that isdb gene sequences were highly conserved, and the recombinant Isdb was successfully expressed. The antibody titer in the immunized groups was increased significantly (P < 0.05) compared with the control, the protective rate of Isdb protein inducted by challenge with the two S. aureus stains Wood46 and HLJ23-1 was 62.5% and 75%, respectively. These results showed that the Isdb protein had high immunogenicity and immunoprotective capacity.

Immunogenicity of Staphylococcus aureus recombinant clumping factor A / 生物工程学报

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein Clumping factor A (ClfA), we amplified clfa genes from S. aureus Newman strain, Wood46 strain and HLJ23-1. The clfa gene from Newman strain was subsequently inserted into pQE-30 vector and the recombinant plasmid was transformed into Escherichia coli strain M15 (pREP4). The recombinant ClfA protein was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level and the concentration of cytokines were measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus Newman, Wood46 and HLJ23-1. These results suggested that clfa gene sequences were highly conserved, and the recombinant ClfA was expressed correctly with good antigenicity. The antibody titer and the concentration of cytokines in the immunized groups increased significantly (P < 0.05) compared with control, and the mice in the immunized groups were protected against the challenge strains to some extent. These results showed that the ClfA had high immunogenicity and immunoprotective potential.

GAPDH activity and immunogenicity of Staphylococcus aureus recombinant GapC protein / 生物工程学报

In order to characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-gamma and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64,000). The concentration of IL-4 and IFN-gamma in GapC groups rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1 x 10(8) CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.

Preparation of two mouse anti-chicken CARP monoclonal antibodies and identification of their biological characteristics / 中国免疫学杂志

Objective:To prepare the monoclonal antibodies against chicken CARP.Methods:The chicken CARP cDNA encoding the N-terminal 110aa length of encoding region was cloned into prokaryotic expression vector pET28b.The BALB/c mice were immunized with purified the recombinant protein,spleen cells of mouse were fused with SP2/0,and the positive clones were screened by indirect ELISA.The properties of McAb were analyzed by ELISA and Western blot.Results:Two hybridomas were proved to have the capability of stably secreting McAb against chicken CARP,designated as 4A8 and 4E6,the subtype of both was IgG1,and the light chain was ?.The titers of ascites were 3.2?104 and 1.28?105 respectively.The Western blot results suggested that they could recognize chicken CARP protein.Conclusion:Two murine hybridomas secreting anti-chicken CARP McAbs have been successfully obtained.