[Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

OBJECTIVE: To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. METHODS: PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. RESULT: lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. CONCLUSION: The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

Diversity of Helicobacter pylori isolates in expression of antigens and induction of antibodies.

AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of H pylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS: The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 H pylori-infected patients, the positive rates of IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases (P=0.016 and P<0.0001, respectively). CONCLUSION: UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.

[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

OBJECTIVE: To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. METHODS: According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. RESULT: The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. CONCLUSION: LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

[Infection status of common intestinal soil-borne nematodes in children from 3 to 6 years old in kindergartens of Hangzhou].

1667 children of 3 to 6 years old were inspected randomly in kindergartens of Hangzhou from April to June 2006. Enterobius vermicularis eggs were detected by cellophane swab technique. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms in fresh stool samples were examined by Kato-Katz thick smear and saturated brine flotation. 216 children were found to have infected with intestinal nematodes (12.96%). The prevalence of E. vermicularis, A. lumbricoides, T. trichiura and hookworm was 4.44%, 8.28%, 0.54% and 0.24% respectively. Higher prevalence has been found in kindergartens with poorer environment and sanitation.

[Membrane location and naturally antibody responses of genus-specific antigen OmpL1s of Leptospira interrogans].

The major aim of this study is to determine the location on outer envelope of the genus-specific antigen OmpL1s of Leptospira interrogans, and the inducement of naturally antibody response and types of the antigen, which will offer the evidences to use OmpL1s as the antigen candidate for developing universal genetic engineering vaccine and detection kit. The serum samples from 156 leptospirosis patients in Sichuan area were detected using microscope agglutination test (MAT). By using PCR plus nucleotide sequence analysis, the genotypes of the dominant L. interrogans serogroups in China were demonstrated. Routine genetic engineering technique was applied to construct the prokaryotic expression systems of genotypes ompL1/1 and ompL1/2, and Ni-NTA affinity chromatography was performed to extract the target recombinant products rOmpL1/1 and rOmpL1/2. Immune aurosol electron microscopy was selected to locate the position of OmpL1s on leptospiral envelope. ELISAs based on rOmpL1s were established to confirm the level and types of specific antibody. The results indicated that L. interrogans serogroup Icterohaemorrhagiae remains to be the most important dominant leptospiral serogroup in Sichuan area. There are two ompL1 genotypes of ompL1/1 and ompL1/2 in the dominant leptospiral serogroup in China. And remarkable differences of the nucleotide and putative amino acid sequence similarities between the two genotypes are present. OmpL1s are the protein molecular that located on the external surface of leptospiral envelope. In the 156 cases of leptospirosis patients' serum samples using different dilutions, the positive rates for rOmpL1/1 or rOmpL1/2 specific IgM are 67.9%-79.5% and 75.0%-75.6%, while for for rOmpL1/1 or rOmpL1/2 specific IgG are 71.8%-79.5% and 75.0%-76.9%, respectively. All the results mentioned above lead to the conclusions that OmpL1s is the leptospiral genus-specific superficial protein antigen of L. interrogans and both rOmpL1/1 and rOmpL1/2 can induce humoral response in individuals naturally infected with L. interrogans as well as produce two serum antibodies IgM and IgG with extensive antigenic-cross reaction. Therefore, rOmpL1/1 and rOmpL1/2 can be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

[Study on the immunogenicity of major leptospiral genus-specific protein antigens and the distribution of antigens in different serogroups of Leptospira interrogans].

OBJECTIVE: The determination of antigenicity and immunogenicity of Leptospira interrogans genus-specific outer envelope proteins (OEPs) will offer evidence for developing universal leptospiral genetic engineering vaccine and detection kit. METHODS: In this study, Ni-NTA affinity chromatography is used to purify the recombinant products rLipL21, rOmpL1/1, rOmpL1/2, rLipL32/1, rLipL32/2, rLipL41/1 and rLipL41/2 expressed by the major genotypes of four leptospiral OEPs of 15 serogroups. SDS-PAGE is applied to examine the expression and purity of the recombinant proteins. Rabbits are intracutaneously immunized with the recombinant proteins to obtain antisera. Microscope agglutination test (MAT) is used to measure the cross inmmunoagglutination titers of antisera. The OMPs of the reference standard strains belonging to 15 serogroups of L. interrogans in China and L. biflexa strain Patoc I are prepared using salt-denature method. By each of the antisera as the first antibody, Western blot assay is established to detect the natural expressions and immunoreactivity of the four OEPs. RESULTS: The outputs of rLipL21, rLipL32/1, rLipL32/2, rLipL41/1l, rLipL41/2, rOmpL1/1 and rOmpL1/2 are 10%, 40%, 35%, 15%, 10%, 30% and 15%, respectively. Each the purified recombinant proteins shows a single fragment after SDS-PAGE. Each the rabbit antisera displays extensive cross immunoreactivity between the products expressed by different genotypes of the same gene and the MAT titers ranging from 1:2-1:128. All the four OEPs can be detectable in the OEPs preparations. However, LipL21 is found to exist only in L. interrrogans. CONCLUSION: The results of this study indicate that all the four OEPs are superficial genus-specific antigens of Leptospira which can be used as the candidate antigens of leptospiral universal vaccine and detection kit.

[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.