Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm / 生物工程学报

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm / 生物工程学报

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization / 中国血吸虫病防治杂志

To prepare and purify the rabbit anti-TgMIC16 polyclonal antibody, so as to apply it in subcellular localization.New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund’s adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescence method.Indirect ELISA showed that the antibody titer was 1∶512 000. Western blotting showed that the recombinant TgMIC16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii.The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.

Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori / 生物工程学报

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.

Immunolocalization of the Signaling Protein l4-3-3 of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To study the localization of the signaling protein 14-3-3 of Schistosoma japonicum (Sj14-3-3)in the parasite. Methods Cercariae were collected from the infected Oncomelania hupensis for the infection of rabbits. Fifteen-day-old schistosomula and adult worms obtained from infected rabbits 15 and 42 days post-infection were used for frozen sections and indirect immunofluorescence staining with monoclonal antibody to rSj14-3-3. Results The results showed that the Sj14-3-3 distributed mainly in the tegument, subtegument, muscle, and parenchyma of both adult worms and 15-day-old schistosomula. Conclusion The wide distribution and large sites of Sj14-3-3 in the parasite were clearly demonstrated, which established a significant clue for further studies of biologic actions and application of 14-3-3 protein.