Non-random arrangement of synonymous codons in archaea coding sequences.

Non-random arrangement of synonymous codons in coding sequences has been recently reported in eukaryotic and bacterial genomes, but the case in archaeal genomes is largely undetermined. Here, we systematically investigated 122 archaeal genomes for their synonymous codon co-occurrence patterns. We found that in most archaeal coding sequences, the order of synonymous codons is not arranged randomly, but rather some successive codon pairs appear significantly more often than expected. Importantly, such codon pairing bias (CPB) pattern in archaea does not seem to completely follow the co-tRNA codon pairing (CCP) rule previously reported for eukaryotes, but largely obeys an identical codon pairing (ICP) rule. Further, synonymous codon permutation test demonstrated that in many archaeal genomes, random mutation alone is unable to cause the observed high level of ICP bias, which strongly indicates that selection force has been involved to shape synonymous codon orders, potentially meeting a global requirement to optimize translation rate.

Uncorrected Preoperative Infection Causing the Death of a Patient with a Thoracic Aortic Aneurysm.

Background: A thoracic aortic aneurysm (TAA) is a known condition seen in cardiovascular practice. A TAA rupture and postoperative infection may result in death. Preoperative infections leading to death are extremely rare. Case Study: A 62-year-old Chinese female was admitted to The First Hospital of Hebei Medical University with a two-day history of abdominal pain. She was diagnosed with a TAA rupture and underwent immediate surgery. The preoperative urine analysis indicated that the positive bacteria and white blood cell count suggested a urinary tract bacterial infection. The patient was administered the empiric antibiotics, cefazolin; however, her blood pressure continued to drop during the perioperative period and she died of uncorrectable acidosis 8 h after the operation. On the second day after death, both the blood and urine cultures were positive for Pseudomonas aeruginosa. Conclusion: Given that this patient with a TAA rupture died of uncorrected acidosis caused by preoperative infection, it is important to evoke the diagnosis in the context of TAA. Routine laboratory indicators are valuable factors for surgeons and physicians in assessing a patient's condition and improving their prognosis.

[Upregulation of FasL/Fas expression and FasL/Fas-associated apoptosis in J774A.1 cells induced by Leptospira interrogans].

OBJECTIVE: To determine the involvement of FasL/Fas pathway in apoptosis of J774A.1 cells induced by Leptospira interrogans. METHODS: The cell infection model was established with mouse monocyte-macrophage J774A.1 cells infected by L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601. The morphological characteristics of apoptotic J774A.1 cells were observed by DAPI staining method, and the apoptosis rate was quantitatively determined by flow cytometry. FasL neutralizing antibody was applied to block the apoptosis. Expression of FasL or Fas in the L.interrogans strain 56601-infected J774A.1 cells was detected by flow cytometry using PE-conjugated monoclonal antibody. RESULT: Chromatin condensation and marginalization were found in J774A.1 cells infected by L.interrogans strain 56601 for 4 h, which became more predominant for 24 h and karyorrhexis was present in some cells. When J774A.1 cells were infected for 4 h and 24 h, the apoptosis rates were 53.6% and 64.31%, respectively. However, the apoptosis rates were decreased to 10.27% and 15.9% after the cells were pre-treated with FasL neutralizing antibody. When J774A.1 cells were infected for 4 h and 24 h, FasL expression rates were increased to 21.69% and 65.70% from that of 4.19% before infection, and Fas expression rates were risen to 91.96% and 88.01% from that of 12.88% before infection. CONCLUSION: Inducement of cell apoptosis is an important mechanism of L.interrogans strain 56601 injuring J774A.1 cells. The strain of L.interrogans is able to up-regulate FasL/Fas expression levels of host cells and induce apoptosis of the cells via FasL/Fas pathway.

[Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups].

OBJECTIVE: To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells. METHODS: The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR. RESULT: mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated. CONCLUSION: The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

[Comparison of serological detection effects of ELISA using rTpN17 or rTpN47 of Treponema pallidum as antigen with that of TPHA and TRUST].

OBJECTIVE: To clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis. METHODS: The whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA. RESULT: The sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive. CONCLUSION: rTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.

[J774A.1 cell apoptosis induced by Leptospira interrogans and effects of caspase-3, -6 activation on apoptosis].

OBJECTIVE: To investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis. METHODS: Mouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively. RESULT: L. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells. CONCLUSION: L. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.

[Re-understanding of liver cirrhosis induced by Schistosomiasis japonica].

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.

Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients.

AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori (H pylori) isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori. METHODS: H pylori strains in biopsy specimens from 157 patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected. The target recombinant proteins rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography. Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody, expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed. RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2% respectively. In the 125 serum samples from the H pylori-infected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4% and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P>0.05, chi2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P<0.01, chi2 = 13.19; P<0.05, chi2 = 6.13). When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P<0.05, chi2 = 5.93; P<0.01, chi2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P<0.01, chi2 = 8.88; P<0.05, chi2 = 5.00). CONCLUSION: The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB, FlaA, HpaA, FlaB, NapA and CagA1, but not VacA.

[Immunoprotective effects of Helicobacter pylori UreB and HpaA bivalence recombinant vaccine with inner adjuvant on experimental infection in mice].

OBJECTIVE: To determine the immunoprotective effects of rUreB and rHpaA bivalence genetic engineering vaccine with inner adjuvant of rLTB on BALB/c mice against H.pylori strain SS1 infection. METHODS: rUreB, rHpaA, rLTB-rUreB-rHpaA, rLTKA63, rLTB and rLTB were collected by NTA-Ni affinity chromatography. Western blot was applied to demonstrate the immunoreactivity of the recombinant protein antigens. Adjuvant activities of rLTB, rCTB and rLTB-rUreB-rHpaA were determined by GM1-ELISA. H.pylori strain SS1-infected BALB/c mouse modal was established to measure immunoprotective effects of different compositions of antigens and adjuvants. H.pylori in gastric biopsy specimens was examined by routine isolation method and silver staining method. Two ELISAs were established to detect specific S-IgA in gastric juices and specific IgA in sera of the immunized mice. RESULTS: rUreB, rHpaA and rLTB-rUreB-rHpaA were recognized by commercial antibody against whole cell of H.pylori and were able to combine to the bovine GM1. The protective rate in the mice immunized with single rUreB or rHpaA was lower than 70%. When using rUreB or rHpaA plus rLTB or rCTB, the positive rates increased to 75.0%-83.3%. With different combination of antigens and adjuvants, the immunoprotective rate of rLTB-jrUreB-rHpaA was as high as 100%, and then was 91.7% for rUreB+rHpaA+rCTB+rLTKA63 and was 90.9% for rUreB+rHpaA+rLTB. Both the rLTB and rCTB showed remarkable effects to induce specific IgA in sera and specific S-IgA in gastric juices of the immunized mice, and the former showed stronger S-IgA-inducing ability than the latter. The positive rates of specific IgA were basically identical to the corresponding mouse immunoprotective rates. S-IgA positive rates specific to rUreB and rHpaA in rLTB-rUreB-rHpaA immunized mice were 100% and 91.7%, respectively. CONCLUSION: The rUreB and rHpaA possess qualified immunoreactivity and antigenicity. The rLTB and rCTB can show adjuvant activity of mucosal immunization. The high immunoprotective rate of Helicobacter pylori UreB and HpaA bivalence recombinant vaccine with inner adjuvant in mice is associated with high dosage of rLTB, larger molecular weight of the antigen and the high levels of local specific S-IgA.

[Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans].

OBJECTIVE: To investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans. METHODS: Ni-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA. RESULTS: Under the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h. CONCLUSION: The expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.

[Construction and identification of the prokaryotic expression system of rLTB/rCTB-rOmpL1/1 fusion genes].

OBJECTIVE: To construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products. METHODS: The fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method. RESULTS: The homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively. CONCLUSION: The fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.

[Apoptosis and ultrastructural lesions in Vero and J774A.1 cells induced by Leptospira interrogans].

OBJECTIVE: To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence. METHODS: A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively. RESULTS: The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis. CONCLUSION: The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

[Phospholipase C activity and alteration of intracellular free Ca2+ levels during internalization of Leptospira interrogans].

OBJECTIVE: To determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans. METHODS: L.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay. RESULTS: The baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05). CONCLUSION: The cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

OBJECTIVE: To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. METHODS: PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. RESULTS: Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. CONCLUSION: An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

[Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene].

OBJECTIVE: To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. METHODS: lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. RESULTS: The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable. CONCLUSION: lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity.

AIM: To clone ureB gene from a clinical isolate of Helicobacter pylori and construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS: ureB gene from a clinical H pylori strain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 H pylori infected patients and to examine rUreB expression in 109 H pylori isolates. RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins. rUreB specifically combined with the commercial antibodies against whole cell of H pylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all H pylori infected patients were positive for UreB antibody and UreB expression were detectable in all tested H pylori isolates. CONCLUSION: A prokaryotic expression system with high expression efficiency of H pylori ureB gene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different H pylori isolates and specific antibody against UreB in sera of H pylori infected patients indicate that UreB is an excellent antigen candidate for developing H pylori vaccine.

Construction of a prokaryotic expression system of vacA gene and detection of vacA gene, VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients' sera.

AIM: To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein, and to determine prevalence of vacA-carrying/VacA expressing H pylori isolates and seroprevalence of specific ant-VacA antibody in H pylori infected patients. METHODS: Polymerase chain reaction technique was used to amplify complete vacA gene of H pylori strain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of H pylori and an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pylori isolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori. RESULTS: In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pylori and to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pylori isolates carried vacA gene, but only 66.1% expressed VacA protein. Of the serum samples tested, 42.4% were positive for specific anti-VacA antibody. CONCLUSION: A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high frequency of vacA gene in H pylori isolates, but with a low frequency of VacA expression and specific anti-VacA antibody in H pylori infected patients implies that VacA is not an ideal antigen for H pylori vaccine.

Helicobacter pylori lipopolysaccharide: biological activities in vitro and in vivo, pathological correlation to human chronic gastritis and peptic ulcer.

AIM: To determine the biological activity of Helicobacter pylori (H. pylori) lipopolysaccharide (H-LPS) and understand pathological correlation between H-LPS and human chronic gastritis and peptic ulcer. METHODS: H-LPS of a clinical H. pylori strain and LPS of Escherichia coli strain O55:B5 (E-LPS) were extracted by phenol-water method. Biological activities of H-LPS and E-LPS were detected by limulus lysate assay, pyrogen assay, blood pressure test and PBMC induction test in rabbits, cytotoxicity test in NIH 3T3 fibroblast cells and lethality test in NIH mice. By using self-prepared rabbit anti-H-LPS serum as the first antibody and commercial HRP-labeled sheep anti-rabbit sera as the second antibody, H-LPS in biopsy specimens from 126 patients with chronic gastritis (68 cases) or gastric ulcer (58 cases) were examined by immunohistochemistry. RESULTS: Fibroblast cytotoxicity and mouse lethality of H-LPS were weaker than those of E-LPS. But the ability of coagulating limulus lysate of the two LPSs was similar (+/0.5 ng/mL). At 0.5 h after H-LPS injection, the blood pressures of the 3 rabbits rapidly declined. At 1.0 h after H-LPS injection, the blood pressures in 2 of the 3 rabbits fell to zero causing death of the 2 animals. For the other one rabbit in the same group, its blood pressure gradually elevated. At 0.5 h after E-LPS injection, the blood pressures of the three rabbits also quickly declined and then maintained at low level for approximately 1.0 h. At 0.5 h after injection with H-LPS or E-LPS, PBMC numbers of the rabbits showed a remarkable increase. The total positivity rate of H-LPS from 126 biopsy specimens was 60.3%(76/126). H-LPS positivity rate in the biopsy specimens from chronic gastritis (50/68, 73.5%) was significantly higher than that from gastric ulcer (26/58, 44.8%) (chi(2)=10.77, P<0.01). H-LPS positivity rates in biopsy specimens from chronic superficial gastritis (38/48, 79.2%) and chronic active gastritis (9/10, 90.0%) were significantly higher than that of the patients with atrophic gastritis (3/10, 30.0%) (chi(2)=7.50-9.66, P<0.01). CONCLUSION: The biological activities of H-LPS were weaker than those of E-LPS, the activities of H-LPS of lowering rabbit blood pressure and inducing rabbit PBMC were relatively stronger. H-LPS may play a critical role in inducing inflammatory reaction in human gastritis.

Establishment of Helicobacter pylori infection model in Mongolian gerbils.

AIM: To establish a stable and reliable model of Helicobacter pylori infection model in Mongolian gerbils and to observe pathological changes in gastric mucosa in infected animals. METHODS: Mongolian gerbils were randomly divided into 18 groups; 6 groups were infected with H pylori clinical strain Y06 (n=6, groups Y), 6 groups were infected with H pylori strain NCTC11637 (n=6, groups N), and 6 uninfected groups as negative controls (n=4, groups C). H pylori suspensions at the concentrations of 2X10(8) and 2X10(9) CFU/mL of strain NCTC11637 and strain Y06 were prepared. The animals in three groups N and in three groups Y were orally challenged once with 0.5 mL of the low concentration of the bacterial suspension. The animals in another three groups N and in another three groups Y were orally challenged with 0.5 mL of the high concentration of the bacterial suspension for 3 times at the intervals of 24 h, respectively. For the negative controls, the animals in six groups C were orally given with the same volume of Brucella broth at the corresponding inoculating time. The animals were killed after 2nd, 4(th) and 6(th) week after the last challenge and the gastric mucosal specimens were taken for urease test, bacterial isolation, pathological and immunohistochemical examinations. RESULTS: Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4(th) and 6(th) week after one challenge were 0%, 16.7% and 66.7%, while in the animals of groups N were 0%, 0% and 16.7%, respectively. Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4(th) and 6(th) week after three challenges were 66.7%, 100% and 100%, while in the animals of groups N were 66.7%, 66.7% and 100%, respectively. In animals with positive isolation of H pylori, the bacterium was found to colonized on the surface of gastric mucosal cells and in the gastric pits, and the gastric mucosal lamina propria was infiltrated with inflammatory cells. CONCLUSION: By using H pylori suspension at high concentration of 2X10(9) CFU/mL for multiple times, the orally challenged Mongolian gerbils can be used as a stable and reliable H pylori infection model. The 2 strains of H pylori can colonize in gastric mucosa of the infected animals and cause mild inflammation reaction.

Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB.

AIM: To demonstrate the effect of lactose as an inducer on expression of the recombinant proteins encoded by Helicobacter pylori ureB and hpaA, and Escherichia coli LTB and LTKA63 genes and to determine the optimal expression parameters. METHODS: By using SDS-PAGE and BIO-RAD gel image analysis system, the outputs of the target recombinant proteins expressed by pET32a-ureB-E.coliBL21, pET32a-hpaA-E.coliBL21, pET32a-LTKA63-E.coliBL21 and pET32a-LTB-E.coliBL21 were measured when using lactose as inducer at different dosages, original bacterial concentrations, various inducing temperatures and times. The results of the target protein expression induced by lactose were compared to those by isopropyl-beta-D-thiogalactoside (IPTG). The proteins were expressed in E.coli. RESULTS: Lactose showed higher efficiency of inducing the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG. The expression outputs of the target recombinant proteins induced at 37 degrees were remarkably higher than those at 28 degrees. Other optimal expression parameters for the original bacterial concentrations, dosages of lactose and inducing time were 0.8, 50 g/L and 4 h for rHpaA; 0.8, 100 g/L and 4 h for rLTKA63; 1.2, 100 g/L and 5 h for both rUreB and rLTB, respectively. CONCLUSION: Lactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG. The results in this study establish a beneficial foundation for industrial production of H pylori genetic engineering vaccine.