RESUMEN
OBJECTIVE: To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein. METHODS: Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis. RESULTS: PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL. CONCLUSION: The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.
Asunto(s)
Catepsina L/inmunología , Fasciola hepatica/enzimología , Fasciola hepatica/genética , Proteínas del Helminto/inmunología , Animales , Catepsina L/genética , Clonación Molecular , Fasciola hepatica/inmunología , Expresión Génica , Cabras , Proteínas del Helminto/genética , Plásmidos , Ratas , Ratas Sprague-DawleyRESUMEN
Bovine leukocyte adhesion deficiency (BLAD) is autosomal recessive disease. The pathogeny of BLAD is genic mutation of CD18-integrins on the leukocyte. In order to know the carrier and occurrence of bovine leukocyte adhesion deficiency (BLAD) among cows age from one to six years old in China, 1,000 cows were investigated by means of amplifying a CD18 gene fragment via reverse transcriptase-PCR followed by restriction digestion with Taq I. Results showed that 19 cows were BLAD carriers, indicating that the BLAD carrier rate was 1.9 percent. In addition, one cow was found to have BLAD.
Asunto(s)
Bovinos/genética , Síndrome de Deficiencia de Adhesión del Leucocito/genética , Síndrome de Deficiencia de Adhesión del Leucocito/veterinaria , Animales , Antígenos CD18/genética , Antígenos CD18/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.