Objective To construct a mutant strain of Nocardia farcinica ( N. farcinica ) IFM10152 with mammalian cell entry 4A gene (mce4A) deletion and to analyze the function of that gene dur-ing infection. -ethods The mutant strain of N. farcinica was constructed through in-frame deletion without antibiotic labeling and verified by PCR and sequencing analysis. To analyze the function of mce4A gene in the interaction between N. farcinica and host cells, in vitro growth experiment, macrophage killing experi-ment using THP-1 ( a human leukemia mononuclear cell line) as the model and adhesion and invasion exper-iments using HeLa cells ( cervical cancer epithelial cells) were carried out. Results The mutant strain with mce4A gene deletion was successfully constructed and named △mce4A. No significant difference in growth rate was observed between the mutant and the wild-type strains. After knocking out the mce4A gene, the ability of N. farcinica to resist macrophage killing was obviously weakened as well as its ability to adhere and invade. Conclusions The mutant strain of N. farcinica with mce4A gene deletion was successfully construc-ted. The mce4A gene might play an important role in the adhesion and invasion of N. farcinica to host cells and its survival in macrophages.

Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.

Objective: To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis. Methods: Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China. Results: The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%. Conclusions The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.

Objective To investigate the effects of high glucose and lysophosphatidylcholine (LPC) on the immune function of in vitro cultured macrophages during Nocardia farcinica infection. Meth-ods RAW264.7 macrophages were cultured in vitro under different conditions as follows: routine culture (control group),50 mmol/L glucose (high glucose group),10 mg/L LPC(LPC groupⅠ),25 mg/L LPC (LPC groupⅡ) and 50 mmol/L glucose+25 mg/L LPC(high glucose and LPC group). The activity of mac-rophages in each group was tested after 6,12,24 and 36 h of culture. After 24 h of culture, macrophages were collected from every group and co-cultured with Nocardia farcinica. Dynamic phagocytosis rates were detected at 1,2,3,4,5 and 6 h after co-culture. Toxic effects of Nocardia farcinica on macrophages and concentrations of IL-10 and TNF-α were measured at 1,3 and 6 h after co-culture. Results Macrophages in all four experimental groups showed decreased activity as compared with those in the control group (P<0.01). Phagocytosis of Nocardia farcinica by macrophages was also reduced by high glucose and LPC. Phagocytosis rates of high glucose group and LPC groupⅡ at 1 and 2 h,LPC groupⅠat 1,2 and 3 h,and high glucose and LPC group at 1,2,3 and 4 h after co-culture were significantly lower than that of the con-trol group (P<0.05 or P<0.01). Compared with the control group, significantly reduced toxic effects on macrophages caused by Nocardia farcinica was observed in the experimental groups (P<0.05 or P<0.01). Compared with the control group,LPC groupsⅠand Ⅱ and high glucose and LPC group had decreased se-cretion of IL-10 at 3 h,and high glucose group and LPC groupⅠhad decreased secretion of TNF-α at 1 h(P<0.05). Conclusion Culture macrophages under the conditions of high glucose and LPC would reduce their activity and impair their ability to phagocytose Nocardia farcinica. Moreover, high glucose and LPC might have impacts on the toxic effects of Nocardia farcinica on macrophages and the secretion of IL-10 and TNF-α.

Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics.Methods A stkP gene knockout (ΔstkP) mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP).The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore).Results S.pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml),but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml,CTX MIC=32 μg/ml).The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.

Objective To express the receptor binding domain (RBD) protein of the Middle East respiratory syndrome coronavirus (MERS-CoV) and to characterize the antigenicity of the purified recombi-nant protein. Methods The codon-optimized gene encoding the RBD protein of MERS-CoV was synthesized and then cloned into the pET30a ( +) vector to construct the recombinant expression plasmid. The trans-formed E. coli BL21 (DE3) strains carrying expression plasmid were induced by IPTG under different condi-tions. The expressed products were purified by using nickel affinity chromatography and further analyzed by SDS-PAGE and Western blot assay. Indirect ELISA was performed to analyze the antigenicity and specificity of RBD proteins expressed in prokaryotic expression systems in human serological test. Results The recom-binant RBD proteins were mainly expressed as conclusion body in an optimal induction condition of 37℃ and 0. 5 mmol/ L IPTG for 4 h. The high purified recombinant RBD proteins were obtained through denaturation and renaturation with a relative molecular mass of about 29×103 . Results of the Western blot assay showed that the recombinant RBD proteins could have specific reaction with the serum samples collected form mice with MERS-CoV infection. Indirect ELISA revealed that the RBD proteins expressed in the prokaryotic ex-pression system showed better sensitivity and specificity in the detection of antibodies against MERS-CoV in human serum samples. Conclusion This study reported the prokaryotic expression and purification of RBD protein of MERS-CoV for the first time, which might pave the way for further investigation on immunological detection of MERS-CoV and development of vaccines against MERS-CoV infection.

Objective To understand how Vibrio vulnificus hemolysin (VvhA) affects the viability of murine liver CD4+ T cells as well as its effects on the numbers of mitochondria and the expression of CD62L. Methods The primary murine liver monocytes (MNs) were isolated from C57BL/ 6 mice and then treated with recombinant VvhA (rVvhA) for 6 hours in vitro. The viability of murine liver CD4+T cells and the expression of CD62L were measured by staining with anti-mouse CD4, CD8, CD44, CD62L and cell via-bility fluorescent dye or fluorescent antibody. Moreover, the cells were simply incubated with MitoTracker or JC-1 probes to label mitochondria and mitochondrial membrane potential, which were further analyzed by using flow cytometry analysis. Results With the increase in the doses of rVvhA, the viability of murine liv-er CD4+T cells was decreased from 81. 5% to 15. 8% . The expression of CD62L on the surface of murine liver CD4+T cells was dramatically decreased. Both the murine liver na?ve and effector CD4+ T cells were sensitive to the cytotoxicity of rVvhA. Moreover, treating murine liver CD4+ T cells with rVvhA resulted in significantly decreased numbers of mitochondria and lower mitochondrial membrane potential. Conclusion The cytotoxicity of rVvhA to murine liver CD4+T cells might be achieved through inhibiting the expression of CD62L, decreasing the numbers of mitochondria and lowering mitochondrial membrane potential.

Objective To clone and express the outer surface protein C ( OspC) from a Chinese Borrelia afzelli FP1 strain and to evaluate the immune protectivity of the recombinant OspC protein ( rOspC) . Methods The gene encoding OspC protein of Borrelia afzelli FP1 strain was amplified by polymerase chain reaction (PCR) and then inserted into pET-30a plasmid to construct the recombinant expression plasmid pET-30a-OspC. The transformed E. coli BL21 strains carrying pET-30a-OspC plasmid were induced by IPTG to express OspC protein. The expressed proteins were purified by Ni-IDA resin chromatography and analyzed by SDS-PAGE and Western blot assay. Indirect immunofluorescence assay ( IFA) was performed to detect anti-rOspC protein antibodies in serum samples from rabbits immunized with rOspC protein. In vitro neutral-ization test was performed for evaluation the immune protectivity of rOspC protein. Results The recombi-nant expression plasmid pET-30a-OspC was successfully constructed and highly expressed in E. coli BL21. A strong antigen-antibody reaction between the rOspC protein and polyclonal antibody against Borrelia afzelli FP1 strain was detected by Western blot assay. The titers of IgG in serum samples from rabbits immunized with rOspC protein were significantly elevated. The in vitro neutralization test indicated that 106/ml of Borre-lia afzelli FP1 strains were neutralized by every anti-OspC protein serum sample from the experiment group. Conclusion The rOspC protein showed a strong immune protectivity against Borrelia afzelli, which could be used in the development of polyvalent subunit vaccine against lyme disease.

Objective To employ Borrelia burgdorferi( B. burgdorferi) , a culturable and genetical-ly transformable spirochete, as a surrogate system to study Treponema pallidum ( T. pallidum) gene function. Methods Bioinformatic analysis revealed that the T. pallidum gene tp0111 encodes the putative sigma factor RpoN. We constructed a B. burgdorferi shuttle vector harboring tp0111. The shuttle vector was then trans-formed into the B. burgdorferi rpoN mutant strain. The phenotype of the resulting B. burgdorferi strain was then determined. Results We successfully constructed the B. burgdorferi rpoN mutant carrying the T. palli-dum gene tp0111. We found that tp0111 could partially complement the B. burgdorferi rpoN mutant. Con-clusion This work provides the first experimental evidence showing that tp0111 is the rpoN gene of T. palli-dum. It also demonstrates that B. burgdorferi can be used as a surrogate system for studying T. pallidum gene function.

Objective To construct a mutant strain of Streptococcus pneumoniae ( S.pneumoniae) with ciaH gene-knockout (ΔciaH) and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations ( MICs ) of penicillin G ( PCN ) and cefotaxime ( CTX ) against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR ( qRT-PCR ) .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1μg/ml) (P<0.01).The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX (P<0.01).Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.