Objective: To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolatedfrom mushroom, against a virulent H9N2 strain in vivo and in vitro. Methods: In trial 1, 50 BALB/c male mi...Objective: To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolatedfrom mushroom, against a virulent H9N2 strain in vivo and in vitro. Methods: In trial 1, 50 BALB/c male mice (8 weeksold) were divided into five groups (n=10 each group) which received a subcutaneous injection of inactivated HgN2(control), inactivated HgN2+0.2% (w/w) alum, inactivated HgN2+0.5 mg recombinant AAL/kg body weight (BW), inac-tivated HgN2+1.0 mg AAl_/kg BW, and inactivated H9N2+2.5 mg AAL/kg BW, respectively, four times at 7-d intervals. Intrial 2, 30 BALB/c male mice (8 weeks old) were divided into three groups (n=10 each group) which received a sub-cutaneous injection of inactivated HgN2 (control), inactivated HmN2+2.5 mg recombinant wild-type AAL (AAL-wt)/kg BWand inactivated H9N2+2.5 mg carbohydrate recognition domain (CRD) mutant AAL (AAL-mutR63H)/kg BW,respectively, four times at 7-d intervals. Seven days after the final immunization, serum samples were collectedfrom each group for analysis. Hemagglutination assay, immunogold electron microscope, lectin blotting, and co-immunoprecipitation were used to study the interaction between hAL and HgN2 in vitro. Results: IgG, IgG1, and IgG2aantibody levels were significantly increased in the sera of mice co-immunized with inactivated HgN2 and AAL whencompared to mice immunized with inactivated H^N2 alone. No significant increase of the IgG antibody level was de-tected in the sera of the mice co-immunized with inactivated HgN2 and AAL-mutR63H. Moreover, AAL-wt, but notmutant AAL-mutR63H, adhered to the surface of H9N2 virus. The interaction between AAL and the H9N2 virus wasfurther demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutininand neuraminidase. Conclusions: Our findings indicated that AAL could be a safe and effective adjuvant capable ofboosting humoral immunity against HgN2 viruses in mice through its interaction with the viral surface glycosylatedproteins, hemagglutinin and neuraminidase.展开更多
C-type lectins are among the most significant pattern recognition receptors(PRRs) found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of...C-type lectins are among the most significant pattern recognition receptors(PRRs) found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn(termed Mj Lectin) from kuruma shrimp Marsupenaeus japonicus was identified. The full-length c DNA of Mj Lectin was 1 245 bp with a 1 011 bp open reading frame(ORF) that encoded a polypeptide of 336 amino acid residues. Mj Lectin consisted of two tandemly arrayed carbohydrate-recognition domains(CRDs), unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2+-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN(Leu134-Pro135-Asn136) in CRD1 and a traditional EPN(Glu299-Pro300-Asn301) in CRD2, respectively. Mj Lectin showed a specific expression pattern in both tissue and cellular levels, for its m RNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus(WSSV) challenge(3.6×108 virions/μL), the expression of Mj Lectin in the hepatopancreas was up-regulated significantly at 48 h(P〈0.01) compared with the control group. These results suggested that Mj Lectin might be involved in the innate immune defense against WSSV infection.展开更多
基金supported by the National Natural Science Foundation of China(Nos.30771501 and 81102850)the National Basic Research Program(973)of China(No.2011CB811302)+2 种基金the National Mega Project on Major Drug Development(No.2009ZX09301-014-1)the Chinese 111 Project(No.B06018)the Wuhan Municipal Project(No.201160923296),China
文摘Objective: To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolatedfrom mushroom, against a virulent H9N2 strain in vivo and in vitro. Methods: In trial 1, 50 BALB/c male mice (8 weeksold) were divided into five groups (n=10 each group) which received a subcutaneous injection of inactivated HgN2(control), inactivated HgN2+0.2% (w/w) alum, inactivated HgN2+0.5 mg recombinant AAL/kg body weight (BW), inac-tivated HgN2+1.0 mg AAl_/kg BW, and inactivated H9N2+2.5 mg AAL/kg BW, respectively, four times at 7-d intervals. Intrial 2, 30 BALB/c male mice (8 weeks old) were divided into three groups (n=10 each group) which received a sub-cutaneous injection of inactivated HgN2 (control), inactivated HmN2+2.5 mg recombinant wild-type AAL (AAL-wt)/kg BWand inactivated H9N2+2.5 mg carbohydrate recognition domain (CRD) mutant AAL (AAL-mutR63H)/kg BW,respectively, four times at 7-d intervals. Seven days after the final immunization, serum samples were collectedfrom each group for analysis. Hemagglutination assay, immunogold electron microscope, lectin blotting, and co-immunoprecipitation were used to study the interaction between hAL and HgN2 in vitro. Results: IgG, IgG1, and IgG2aantibody levels were significantly increased in the sera of mice co-immunized with inactivated HgN2 and AAL whencompared to mice immunized with inactivated H^N2 alone. No significant increase of the IgG antibody level was de-tected in the sera of the mice co-immunized with inactivated HgN2 and AAL-mutR63H. Moreover, AAL-wt, but notmutant AAL-mutR63H, adhered to the surface of H9N2 virus. The interaction between AAL and the H9N2 virus wasfurther demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutininand neuraminidase. Conclusions: Our findings indicated that AAL could be a safe and effective adjuvant capable ofboosting humoral immunity against HgN2 viruses in mice through its interaction with the viral surface glycosylatedproteins, hemagglutinin and neuraminidase.
基金The National High-Technology R&D Program(863 Program) of China under contract No.2012AA10A409China Agriculture Research System under contract No.CARS-47
文摘C-type lectins are among the most significant pattern recognition receptors(PRRs) found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn(termed Mj Lectin) from kuruma shrimp Marsupenaeus japonicus was identified. The full-length c DNA of Mj Lectin was 1 245 bp with a 1 011 bp open reading frame(ORF) that encoded a polypeptide of 336 amino acid residues. Mj Lectin consisted of two tandemly arrayed carbohydrate-recognition domains(CRDs), unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2+-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN(Leu134-Pro135-Asn136) in CRD1 and a traditional EPN(Glu299-Pro300-Asn301) in CRD2, respectively. Mj Lectin showed a specific expression pattern in both tissue and cellular levels, for its m RNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus(WSSV) challenge(3.6×108 virions/μL), the expression of Mj Lectin in the hepatopancreas was up-regulated significantly at 48 h(P〈0.01) compared with the control group. These results suggested that Mj Lectin might be involved in the innate immune defense against WSSV infection.
基金supported by grants from National Basic Research Program of China(30800170)The Special Project on Drug Discovery from The Ministry of Public Health of China(2009ZX09103-676)~~