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Objective To construct a prokaryotic expression vector of Salmonella paratyphi A ompA gene, and to determine immunogenieity and immonuprotection of the recombinant expressed product rOmpA and carrying frequency of ompA gene in S. paratyphi A isolates. Methods ompA gene was amplified from a clinical S. paratyphi A strain JH01 by PCR, cloned into T-A cloning vector, and then the prokaryotic expression vector was constructed. The rOmpA expression was determined by SDS-PAGE. Antigenicity and immunoreactivity of rOmpA were determined by immunodiffusion test, Micro-Widal's test and Western blot assay, ompA gene exiting in 98 S. paratyphi A isolates was detected by PCR. By using immunoprotective effect of rOmpA against the challenge of S. paratyphi A strain 50001 in mice were determinded. Results Both the nucleotide and putative amino acid sequence identities of the cloned ompA gene were 100% com-pared to the reported corresponding sequences. The expression yield of rOmpA was approximately 65% of the total bacterial proteins, rOmpA was able to induce specific antibody in rabbits, and reacted efficiently with rabbit antisortm or the antiserum against whole cell of S. paratyphi A detected by Western blot. 94.9% (93/98) of the S. paratyphi A isolates had ompA gene. 41.7% (5/12) and 58.3% (7/12) of the mice im-murtized with 100 and 200 μg rOmpA were survival after lethal challenge with S. paratyphi A strain 50001. The titer of antibody to the H antigens of S. paratyphi spp in the sera from rOmpA immunized mice and sur-viral mice in the S. paratyphi A challenge detected by Micro-Widal's test was 1:5-1:40. Conclusion ompA gene has an extensive distribution in clinical isolates of S. paratyphi A. rOmpA possesses an immunogenicity and a certain immunoprotective effect, and can be used as the candidate antigen in genetic engineering vaccine.
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Objective To clone mcp1, mcp2 and mop3 genes that encoding methyl-accepting chemotaxis proteins(MCP) of Campy/obacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and chemotactic inducers. Methods The segments of mep1, mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning. Prokaryofic expression systems of the genes were subsequently constructed. SDS-PAGE pins Bin-Rod Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1, rMCP2 and rMCP3, and Ni-NTA affinity chromatography was performed to purify the rMCPs. Rabbits were immunized with each of the three rMCPs to obtain antisera. Immunodiffusion assay was performed to measure the titers of antisera. IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography, and IgG F(ab')2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactic model in vitro of C. jejuni based on HAP( hard-agar plus) method was established to determine the chemo-taxis-inducing effect of eight candidate substances. Chemotaxis inhibition test based on IgG F(ab')2 bloc-king was applied to determine the function and diversity of MCPs. Results The segments with expected si-zes amplified from mcp1, racp2 and mcp3 genes were obtained by PCRs, and their nucleotide and putative amino acid sequences were completely same as the reported. The constructed prokaryotic systems could effi-ciently express rMCPs with the yields about 10% of the total bacterial proteins. Immunization with rMCP1, MCP2 and rMCP3 enables the rabbits to produce specific antibody. All the antisera had 1: 4 immunodiffusion titers. Both bovine bile and sodium deoxycholate(DOC) were able to induce ehemotactie movement of C. je-juni in a dosage-dependent manner ( P < 0.05 ). When MCP1 and MCP2 were blocked with their IgG F(ab')2, the ehemotaetic ability of C. jejurd were remarkably decreased (P <0. 05). However, MCP3 blocking did not affect the chemotaxis ( P > 0.05 ). Conclusion The C. jejuni MCPs are successfully ex-pressed in this study. Bovine bile and DOC are the inducers for chemotaxis of C. jejuni, and MCP1 and MCP2 are involved in the process of ehemotaxis iadueed by DOC.
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Objective To determine the effect of Mycobacterium tuberculosis spp. inducing transformation of THP-1 cells to epithelioid cells (EC) and the involved signaling pathways and their regulations. Methods THP-1 cells infection models respectively infected with M. tuberculosis strains H37Rv, bovis and phlei were established. Indirect immunofluorescent staining assays were used to detect the expressions of monocyte/macrophage differentiation antigen CD115 and EC differentiation antigen CD82 of the THP-1 cells before or after infection. By Sandwich ELISA Kits, the phosphorylation levels of p38MAPK, Akt1 and STAT3 of the THP-1 cells before or after infection were measured. The alterations of CD115 and CD82 expression levels were examined when the associated signaling pathways were blocked with specific blocking agents. Results CD115 expression was weakened and CD82 expression was strongly increased in all the THP-1 cells infected with the three strains. A temporal up-regulation of the p38MAPK phoshporylation level but no obvious alteration of Akt and STAT3 phosphorylation levels after THP-1 cells infected by strain H37Rv or bovis. The THP-1 cells infected with anyone of the three strains continuously expressed CD115 after MAPK, PI3K/Akt or JAK/STAT of the cells was blocked. Although JAK/STAT was blocked, the THP-1 cells respectively infected with the three different strains still expressed CD82. However, CD82 expressed in THP-1 cells infected by the strain H37Rv or bovis was disappeared when p38MAPK and PI3K/Akt pathways of the cells were blocked. Conclusion Strain H37Rv and bovis can induce the infected THP-1 cells transforming to EC and p38MAPK and PI3K/Akt signaling pathways participate and regulate the transformation procedure. Of the two signaling pathways p38MAPK seems to be more important.
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Objective To screen the efficient antigenic epitopes in genus-specific envelope proteins OmpL1 and LipL21 of Leptospira interrogans for further development of multiple antigenic peptide (MAP)vaccine.Methods Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in OmpL1 and LipL21 molecules were screened.Nucleotide fragments of each epitopes were amplified by PCR and then constructed their phage display systems.Using antisera against rOmpL1,rLipL21,L.interrogans serogroup Icterohaemorrhagiae strain Lai and leptospirosis patients' sera as the first antibodies.respectively,Western blot assays were performed to determine the immunoreaetivity and reactive ability of the epitopes with different antisera.Resuits Four combined epitopes of OmpL1 and two combined epitopes of LipL21 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢ protein and successfully expressed.All of the antisera could recognize each of the epitopes.Based on the results of Western blot,the two LipL21 epitopes at 97-112 and 176-184 showed similar strong hybridization signals with any of the antisera,and the hybridization signals of four OmpL1 epitopes with the three antisera were 173-191,87-98,297-320 and 59-78,from strong to weak.Conclusion The six combined epitopes in this study are efficiently antigenic.And the epitopes at positions 97-112 and 176-184 in LipL21 as well as the epitopes at position 87-98 and 173-191 in OmpL1 have a potential for developing leptospiral MAP vaccine.
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Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.
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Objective To construct the multiple antigenic peptide (MAP) gene and E. coli ex-pression system, based on the out membrane protein OmpL1, LipL21 and LipL32 from Leptospira interro-gans, and better understanding of the immunological activity of the recombinant protein. Methods Using MI3KE display and Western blot, the advantage epitopes of OmpL1, LipL21 and LipL32 were identified and used to synthesize a new gene, then its prokaryotic expression system was constructed. The expression of re-combinant protein was determined by SDS-PAGE. The immunity activity of the recombinant protein was iden-tified by Western blot and ELISA. Results The synthetic gene was effectively expressed in E. coli and mainly presented in soluble form. The expression protein could react with the antileptospirosis antibodies in rabbit and human sera, which contained different serogroups. Conclusion The recombinant MAP gene of leptospires was successfully constructedand and expressed in E. coli. The recombinant protein had a good immune activity, and could cross-reacted with antileptospirosis antibodies from different serogroups.
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To reconstruct the heat-labile enterotoxin subunit B (LTB) gene of Escherichia coil in order to increase the outputs of the prokaryotic expression on recombinant LTB (rLTB), and to determine its immune adjuvant activity on mucosa,the nucleotide sequence of the whole length of LTB gene was synthesized according to the preferred codons of E. coli, and the prokaryotic expression plasmid pET32a-rLTB and its expression system in E. coli BL21DE3 were reconstructed. The recombinant plasmid was extracted and the inserted sequence of rLTB gene was determined. Meanwhile, the expression quantity of the reconstructed rLTB was identified by SDS-PAGE and BioRad agarose image analyzing system, and compared with that of the un-reconstructed rLTB. The abilities of the reconstructed rLTB and the un-reconstructed rLTB to bind with bovine GM1 were determined by means of GM1-ELISA assay. By using the recombinant urease subunit B as antigen, the effects of the reconstructed and the un-reconstructed rLTB on the improvement of immune protection of BALB/c mice infected with Helicobacter pylori strain SS1 and the induction of S-IgAs in infected mice were assayed. The experimental results showed that the expression quantity of the reconstructed rLTB approached upto 35.4% of the total bacterial proteins after induction with 1 mmol/L IPTG for pET32a-rLTB-E. coliBL21DE3 and to be 12.6 times higher than that of the un-reconstructed rLTB (2.8 %). In addition, both the abilities of the recombinant reconstructed LTB and the un-reconstructed rLTB to bind with bovine GM1 could be demonstrated by GM1-ELISA. The immune protection rate of the recombinant urease subunit B in the infected mice was 66.7%; and it could reach up to 91.7% with a significantincrease of the specific S-IgA level, when it was immunized with the reconstructed or the un-reconstructed rLTB. It is concluded that the reconstructed LTB gene in the present study shows a remarkable increased outputs of expression of this gene with a strong immune adjuvant activity on mucosa.
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AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N. gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N. gonorrhoeae were amplified by using high fidelity PCR. The target amplification fragments were sequenced after T-A cloning. Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed. A prokaryotic expression system of PIA gene was constructed. Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA. rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962), the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%, respectively, which indicated that all the isolates were belonging to serovars IA6. Output of rPIA was as high as 50.1% of the total bacterial proteins. The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N. gonorrhoeae isolates and sequences of the encoding gene are relatively conserved. The constructed prokaryotic expression system is able to express rPIA with high efficiency, which may lay a foundation for further development of serological detection kit and vaccine of N. gonorrhoeae.
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Whether the hepatic pipestem fibrosis induced by schistosomiasis japonica can result in cirrhosis,confusion exists among parasitologists in China.Evidence from national and international pathologists and clinicians confirmed that the pipestem fibrosis could develop into cirrhosis undoubtedly.Owing to different pathogenic causes,the characters of cirrhosis are different.To re-understand cirrhosis induced by schistosomiasis japonica is of significance for the diagnosis and treatment of the advanced patient.
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AIM:To construct a prokaryotic expression system of staphylococcal enterotoxin A(SEA)gene and determine the effects of the recombinant expression product rSEA in promoting lymphocyte proliferation and inhibiting tumor cell growth.METHODS:PCR was used to amplify entire SEA gene of S.aureus strain ATCC13565.The cloned SEA gene was sequenced after T-A cloning.SDS-PAGE was applied to measure the output of rSEA expressed by pET32a-SEA-E.coli BL21DE3.Ni-NTA affinity chromatography was performed to extract rSEA.Cytotoxicity of rSEA to Vero cells was detected using TCID_ 50 titration method and then the value of TCIC_ 50 was determined.MTT colorimetry was established to examine the effects of rSEA at different dosages on proliferation of mouse splenocytes and human peripheral blood mononuclear cells(PBMC)as well as on growth of HepG2 cells and HeLa cells in vitro.RESULTS:In comparison with the published corresponding sequences,similarities of the nucleotide and putative amino acid sequences of the cloned SEA gene were 100%.The output of rSEA was approximate 25% of the total bacterial proteins.rSEA had a cytotoxicity with TCIC_ 50 of 3.14 ?g to Vero cells.1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC(P