[Cloning and expression of rotavirus SA11 VP7 and preparation of IgY antibodies against recombinant VP7].

OBJECTIVE: To prepare eukaryotic expression of rotavirus (RV) SA11 capsid protein VP7, and to generate and purify yolk immunoglobulin (IgY) antibodies against the recombinant VP7 from Roman hens. METHODS: MA104 cells were infected with the standard SA11 strain and the culture fluid was collected. A DNA fragment of 978 bp encoding SA11 VP7 was obtained by RT-PCR amplification from genomic RNA of RV SA11. The PCR products were ligated to pMD18-T vector following the confirmation by DNA sequencing and sub-cloned into pPICZalphaB. The recombinant pPICZalphaB-SA11 VP7 was transformed into E coli Top10. The plasmids were linearized by digestion of BstXI and transformed into Pichia pastoris X-33 through electroporation by DNA sequencing. The transformants were induced with methanol for expression. The cultural supernatant was subjected to SDS-PAGE and Western blotting. Fusion expression was purified through the column of affinity chromatography. IgY was identified and purified by SDS-PAGE and Western blotting from eggs of Roman hens immunized with recombinant SA11 VP7. RESULTS: The RNA extracted from the RV culture fluid consisted of 11 bands visualized by silver staining. The expression vector pPICZalphaB-SA11 VP7 was constructed and the fusion protein in Pichia pastoris X-33 was harvested and purified. The recombinant SA11 VP7 with molecular weight of 40 200 was identified by Western blotting. The IgY antibodies against the recombinant SA11 VP7 were produced with a purity of 95 percent and yield of 10.2 mg per egg. CONCLUSION: The preparation of IgY antibodies to recombinant SA11 VP7 might lay a foundation for the development of vaccines and diagnostic techniques.

Application of signaling protein 14-3-3 and 26 kDa glutathione-S-transferase to serological diagnosis of Schistosomiasis japonica.

Schistosomiasis japonica is currently one of the most serious parasitic diseases and over 670000 people are infected in China by the end of 2006. In order to establish an effective diagnostic method, the gene coding for Sj14-3-3 and Sj26kDa GST were cloned and expressed separately in Escherichia coli as fusion protein with His-tag. The rSj14-3-3 and 26kDa rSjGST were combinedly used as antigens for enzyme-linked immunosorbent assays (ELISA) to diagnose acute and chronic S. japonica. Our results showed that the sensitivity in diagnoses of both acute and chronic schistosomiasis was 94.4% (67/71) and 80.7% (96/119), respectively. The specificity was 94.7% applying 132 sera from people living in S. japonicum-free areas. The data also showed that the recombinant proteins cross-react with Clonorchis sinensis and hookworms at a rate of 11.8% and 5.3% respectively. Parallel tests were conducted among ELISA, indirect hemagglutination assay (IHA) and circular ovum precipitin test (COPT) to determine anti-S. japonicum antibodies in sera of patients with schistosomiasis, healthy control, and those infected with other parasites and the results showed no significant difference in sensitivity for acute schistosomiasis between ELISA and IHA assays (chi(2)=1.33, P>0.05), but significant between ELISA and COPT assays (chi(2)=6.72, P<0.01). Our results also revealed significant difference in positive rate between ELISA and IHA (chi(2)=24.74, P<0.005), as well as between ELISA and COPT (chi(2)=58.14, P<0.005). These results suggest that the rSj14-3-3 and r26kDa SjGST would be effective diagnostic antigens for detection of antibodies to S. japonicum in human. Due to the easy production, high sensitivity and specificity, the recombinant proteins tested in this study can be considered as candidate reagent for immunological diagnosis of human schistosomiasis.

[Amiota magna as an intermediate host of Thelazia callipaeda under laboratory conditions].

A number of flies around the eyes of a person or around a fruit bait were collected from Huangshan Mountain, and experimentally infected by newborn larvae of Thelazia callipaeda. After 20 days, the flies were examined for T. callipaeda. Following dissection, 3 (30%, 3/10) of Amiota magna, and 55 (21.6%, 55/255) of A. okada were found infected by T. callipaeda. The susceptibility of T. callipaeda is similar in the two species fruit flies (chi2=0.0584, P> 0.05). The rabbits were infected by infective larvae of T. callipaeda from A. magna. At the 35th day after infection, the newborn larvae and worms of T. callipaeda were found in the conjunctival sac of rabbits. This study suggested that A. magna acts as intermediate host of T. callipaeda under laboratory conditions.