[Gene cloning, expression and serological evaluation of diagnostic antigen Em18 for alveolar echinococcosis].

OBJECTIVE: To clone, express and serologically evaluate the Em18 antigen gene of Echinococcus multilocularis for diagnostic purpose. METHODS: Polymerase chain reaction (PCR) was employed for amplification of the target gene fragments which was then ligated with pET28a+ vector. The constructed plasmid was transferred into E. coli BL21 (DE3) for expression. The recombinant proteins were purified with Ni-NTA agarose by affinity chromatography. 237 sera were used for evaluating diagnostic value of the recombinant Em18 antigen. RESULTS: Two high-level expression clones (designated as ReEm18-1 and ReEm18-2) were obtained. ReEm18-1 showed the expected sequence, ReEm18-2 showed the same sequence but with 27 nucleotides deletion. The molecular weight of the two expression proteins was Mr 28,000 and 26,000, respectively. Serological evaluation by ELISA was carried out using sera from 101 patients with alveolar echinococcosis (AE), 47 with cystic echinococcosis (CE), 30 with cysticercosis (CC), 10 with hepatic cancer (HC), 9 with schistosomiasis (Sj) and 40 from healthy persons (NH) from both endemic and non-endemic areas. The results showed an overall sensitivity of 86.1% and 90.1% with ReEm18-1 and ReEm18-2 for AE sera, specificity 93.4% and 94.1%, positive predictive value 90.6% and 91.9%, negative predictive value 90.1% and 92.8% and efficiency 90.3% and 92.4%, respectively. The correlation analysis between the size of AE lesions and the serum absorbance reacted with recombinant Em18 antigens showed that there was a positive correlation between antibody level and the course of the disease. CONCLUSION: ReEm18 antigens are specific for AE diagnosis, and the serum antibody level displays a good correlation with the course of the disease at early stage. Similar results achieved by both ReEm18-1 and ReEm18-2 antigens.

Comparison of mitochondrial cytochrome oxidase 1 DNA sequences from Necator americanus hookworms maintained for 100 generations in golden hamsters (Mesocricetus auratus) and hookworms from natural human infections.

The human hookworm Necator americanus was maintained through one hundred generations in the golden hamsters. The strain is now routinely maintained in laboratory hamsters through serial passage, and is the laboratory strain of choice for vaccine studies. Comparison of the mitochondrial cytochrome oxidase 1 (cox-1) sequences was shown previously to be useful for comparing the genetic structure of populations of N. americanus in China. Cytochrome oxidase 1 genes were amplified by the polymerase chain reaction, and the sequences compared to those of N. americanus recovered from infected humans from several regions in China. Sequence comparison revealed little difference between the laboratory strain and the field isolates at the cox-1 locus, but also indicated that the laboratory strain is represented by a single cox-1 haplotype. These results suggest that the laboratory strain of N. americanus has undergone a severe genetic bottleneck, and that the genetic diversity in other genes, including potential vaccine antigens, could be similarly limited.

[Western blotting analysis of specific antigens from different components of Echinococcus metacestodes].

OBJECTIVE: To analyze antigens for searching specific antigenic components for immunodiagnosis of echinococcosis. METHODS: Fourteen crude antigens from different tissues (cyst fluid, protoscoleces, laminated layer and germinal layer) of Echinococcus granulosus and E. multilocularis metacestodes and other 4 species of cestodes were analyzed by Western blotting. The differences of protein bands were compared for the 14 crude antigens by reacting with pooled sera from cystic echinococcosis (CE) and alveolar echinococcosis (AE) patients. RESULTS: Eleven protein bands from the antigens reacted nonspecifically with sera from both CE and AE patients were Mr 130000, 100000, 94000, 80000, 75000, 66000, 62000, 52000, 38000, 32000, 24000. The highly specific protein bands recognized by AE sera were Mr 120000, 109000, 86000, 59000, 43000, 28000, 20000, 18000, and by CE sera were Mr 41000, 40000, 22000, 16000 and 12000. CONCLUSION: Different antigens shared by the two species of Echinococcus were examined and potential antigenic proteins specific for AE or CE sera were found, providing useful information for further identifying specific antigens for immunodiagnosis.

[Polymorphism analysis of cytochrome C oxidase 1 (COI) gene in Necator americanus collected from 5 provinces in China].

OBJECTIVE: To detect and compare the COI gene sequences of Necator americanus collected from the provinces of Sichuan, Hainan, Yunnan, Hubei and Jiangsu, and to analyze the genetic diversity of the geographic isolates. METHODS: COI genes of N. americanus were amplified from the genomic DNA by PCR and sequenced. RESULTS: The COI gene sequences of N. americanus from five provinces were 97%-99% identical over 595 bp, and base variation occurred in 19 nucleotide sites in which the transition was more frequent than the transversion. The difference between the sequences ranged from 1.34% to 2.18%. CONCLUSION: The COI gene sequences show high identity among the geographic isolates of N. americanus with some difference at specific nucleotide sites.

[Cloning and expression of specific antigen genes of Ancylostoma caninum].

OBJECTIVE: To search for the gene encoding specific antigen of Ancylostoma caninum that induces host's protective immunity. METHODS: A lambda ZAP II cDNA library of A. caninum was screened with sera from dogs immunized subcutaneously with hookworm larvae(L3). After sequencing, insert gene (AcAg) from positive clones was ligated into PUC18 and PET28C. Recombinant pET28C plasmid was induced to expressed protein in the E. coli BL21. The characteristic of recombinant protein is analyzed by SDS-PAGE and Western blotting assay. RESULTS: Five positive clones were obtained, and proved to be the same. The insert gene (AcAg) in pET28C vector expressed a recombinant protein of about 43 kDa. Using Western blotting assay, this protein was recognized by the sera from dog immunized with Ancylostoma caninum third-stage infected larvae and used for screening library. CONCLUSION: The AcAg, which exhibits 35% homologous to Caenorhabditis elegans gene unc-89, is a novel specific antigen of A. caninum. Its ability to elicit the protective immunity and the probability of the recombinant protein as a vaccine need to be further evaluated.

[Reexamination of specific antibodies in sera of cystic echinococccosis patients with IgG negative seroresponse].

OBJECTIVE: To explore the factors of false negative antibody response in patients with echinococcosis granulosus (Eg) for improving immunodiagnosis. METHODS: Indirect ELISA and sandwich ELISA were used to detect the specific antibody of IgG subclass (IgG1, IgG2, IgG3, IgG4) and IgA, IgM, IgE, as well as circulating antigen (CAg) and immunocomplex (CIC) in sera of Eg patients with negative response of total IgG. RESULTS: Among 42 sera with IgG negative seroresponse, 32 were positive with IgG subclass, IgA, IgM or IgE antibody, 10 were negative in all 7 kinds of antibody response. The detection rate of specific IgG1, IgG4, IgA, IgM and IgE was 42.9%, 11.9%, 28.6%, 26.2% and 21.4% respectively, being significantly higher than in sera of the control. IgM level in children was higher than that in adults. IgG subclass in patients with liver Eg was higher than that of pulmonary hydatidosis, when testing with IgG1 combined one or two of the other six Ig antibodies. The highest positive rate (64.3%) was seen in IgG1 + IgA + IgM antibody system. The positive rate of CAg and CIC in IgG negative patients was 28.57% and 30.95%, respectively. CONCLUSION: The factors involved with seronegative response of total IgG in Eg patients might be low specific IgG, variant Ig antibody expression and formation of CIC. Combined detection of IgG1 + IgA + IgM could enhance the sensitivity of serological tests in Eg patients.