Preparation of rabbit polyclonal antibody against porcine gasdermin D protein and determination of the expression of gasdermin D in cultured cells and porcine tissues
Gasdermin-D (GSDMD) is a member of the gasdermin (Gsdm) protein family, and its cleavage by inflammatory cysteine proteases (caspases, CASPs) is a critical event in cell pyroptosis. The role and functions of GSDMD on mice and humans are widely studied, but its expression, structure, and function in other species are less known.
In the present work, rabbit anti-porcine GSDMD (pGSDMD) polyclonal antibody was prepared by immunizing New Zealand white rabbits with prokaryotic expressed recombinant pGSDMD (rpGSDMD). The prepared polyclonal antibody showed good specificity in Western blot and indirect immunofluorescence (IIF) assays.
Western blot results showed that the polyclonal antibody could recognize overexpressed pGSDMD in human embryonic kidney cells (HEK293T) and endogenously expressed pGSDMD in cultured intestinal porcine enterocytes (IPEC-J2) and porcine kidney cells (PK-15). Western blot also revealed that pGSDMD was expressed in the heart, liver, lung, kidney, gallbladder, and jejunum of pigs. HEK293T cells overexpressing GSDMD showed green fluorescence in the IIF assay only after being treated with 0.3% Triton-X 100, which indicated that the full-length pGSDMD was located in the plasma but not on the cell membrane. This work provides a useful tool and basic information for further studies on pGSDMD.
Preparation and identification of rabbit polyclonal antibody against BCR-ABL b3a2 fusion protein
Objective To prepare and identify rabbit anti-breakpoint cluster region-Abelson leukemia virus oncogene (BCR-ABL) b3a2 subtype polyclonal antibody. Methods A peptide containing the fusion sequence of the b3a2 subtype BCR-ABL fusion protein was designed and synthesized with the purity higher than 90%. The fusion polypeptide was coupled to Keyhole Limpet hemocyanin (KLH) and used to immune New Zealand rabbits. Antiserum was purified after multiple immunizations, in addition to using the b3a2 subtype fusion polypeptide for affinity purification.
Peptides harboring only BCR or c-ABL amino acid sequences were also synthesized and used to purify the antibody in the secondary purification. The antibody that only bound to part of the epitope was absorbed and removed. ELISA and Western blotting were performed to determine the antibody titer and specificity.
Results The rabbit serum background was low before immunization. The titer of the polyclonal antibody reached 1:32 000 after immunization, which met the experimental requirements. Western blotting showed that the antibody could specifically recognize the b3a2 subtype fusion protein of BCR-ABL. Conclusion The experiment has prepared the specific rabbit polyclonal antibody against BCR-ABL b3a2 subtype.
Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies
High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) – a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed.
The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage.
Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.
Prokaryotic expression of mouse Stra8 and preparation and identification of rabbit anti-mouse Stra8 polyclonal antibody
Objective To prokaryotically express mouse stimulated by retinoic acid gene 8 (Stra8) and prepare rabbit anti-mouse Stra8 polyclonal antibody. Methods The recombinant plasmid pET28a-Stra8 was constructed by cloning technology and identified by double enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression of Stra8 recombinant protein was induced by IPTG. The prokaryotic protein was purified by His-TAG affinity chromatograph and then used to immune New Zealand white rabbits to obtain Stra8 polyclonal antibody.
ELISA, Western blot and immunofluorescence assays were used to determine the antibody titer, validity and specificity, respectively. Results The recombinant plasmid pET28a-Stra8 was constructed successfully as double enzyme digestion and sequencing showed, and the prokaryotic protein was expressed and purified effectively. The titer of the polyclonal antibody reached 1:106. Western blotting showed that the polyclonal antibody could specifically recognize native Stra8 protein in the testis. Immunofluorescence assay revealed that the polyclonal antibody had good reactivity, and could recognize Stra8 protein in mouse testis. Conclusion Stra8 prokaryotic protein can be effectively induced in E. coli and specific rabbit anti-Stra8 polyclonal antibody has been prepared.
Prokaryotic expression of PE8 protein from Mycobacterium tuberculosis (H37Rv) and preparation of its polyclonal antibody in rabbits
Objective To clone proline-glutamate 8 (PE8) gene segment from Mycobacterium tuberculosis (H37Rv), construct the recombinant plasmid pET28a-PE8, express recombinant PE8 protein, and prepare its polyclonal antibody. Methods Using a standard homologous recombination cloning technology, we cloned the PE8 gene into the prokaryotic vector pET28a. After sequence confirmation, it was transformed into E. coli BL21 (DE3) and treated with 0.5 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) to induce protein expression. We purified and renatured the recombinant PE8 protein, and immunized New Zealand rabbits to prepare the polyclonal antibody. Antibody titer was determined by indirect ELISA and the specificity was evaluated by Western blot analysis.
Results The recombinant plasmid pET28a-PE8 was successfully constructed, and the PE8 protein was primarily expressed in an inclusion body in E. coli. After renaturation and purification, a purity of about 90% of the recombinant protein was achieved. The titer of the polyclonal antibody was higher than 1:430 080. The polyclonal antibody could specifically recognize the recombinant PE8 protein. Conclusion We have successfully expressed and purified recombinant PE8 protein, which can be further utilized to generate PE8 polyclonal antibody with acceptable titer and specificity.