The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Female ; Animals ; Zona Pellucida Glycoproteins ; Membrane Glycoproteins/metabolism* ; Chickens/genetics* ; Egg Proteins/metabolism* ; Receptors, Cell Surface ; Estrogens

OBJECTIVETo study the influence of heat-induced epitope retrieval (HIER) on endogenous avidin-binding activity (EABA) and to establish an effective way to block EABA in immunohistochemistry.

METHODSSystematically screening EABA in 164 (679 samples) formalin-fixed and paraffin-embedded human tissues including 76 (102 samples) normal tissues and 88 (577 samples) tumor tissues as well as 4 (80 samples) formalin-fixed and paraffin-embedded rat normal tissues using tissue array (tissue chip), HIER, immunohistochemistry and egg white solution blocking. In addition, EABA was also examined in 9 (15 samples) human frozen tissues.

RESULTS(1) EABA was detected in frozen tissues. (2) No staining for EABA was seen in formalin-fixed and paraffin-embedded tissues. (3) EABA was revealed after the tissues treated with microwave HIER. (4) The density of signal for EABA was variable from tissue to tissue and cell to cell. (5) The signals of EABA expressed in scatter or diffuse in tissues and in granular form in cytoplasm. (6) EABA was found in a wide range of epithelial tissues, especially in gland epithelia of normal and tumor tissues. These included kidney, adrenal cortex, liver, C cells of thyroid gland, oxyphil cells of parathyroid, fundal gland of stomach, sebaceous gland of skin, duct of salivary; oncocytoma and papillary adenocarcinoma of kidney and thyroid gland, adenolymphoma of parotid, carcinoma of liver cell, adenocarcinoma of stomach, colon, prostate, gall bladder and endometrium, and so on. (7) EABA was easier revealed by higher pH value buffer (EGTA pH 9.0) than that with lower pH value (EDTA pH 8.0 and citrate pH 6.0). (8) The revealed EABA could be effectively blocked using 20% egg white solution.

CONCLUSIONSHIER could unmask EABA in formalin-fixed and paraffin-embedded tissues. The unmasked EABA present in a wide range of human normal and tumor tissues as well as in rat normal tissues. The EABA could influence routine immunohistochemistry staining when using (strept)avidin-horseradish peroxidase detective system. The egg white solution could effectively block EABA and eliminate the influence of EABA on immunohistochemistry.

Animals ; Avidin ; antagonists & inhibitors ; metabolism ; Biotin ; metabolism ; Cells, Cultured ; Egg Proteins ; pharmacology ; Epithelial Cells ; metabolism ; Epitopes ; Female ; Hot Temperature ; Humans ; Immunohistochemistry ; Male ; Neoplasms ; metabolism ; pathology ; Rats

OBJECTIVETo investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.

METHODSThe recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.

RESULTSThe recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.

CONCLUSIONIt is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.

Baculoviridae ; genetics ; metabolism ; Blotting, Western ; Egg Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; methods ; Zona Pellucida Glycoproteins

Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.
Blotting, Western ; Chromatography, Affinity ; Egg Proteins ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism ; Zona Pellucida Glycoproteins

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

OBJECTIVETo investigate the histomorphology and the expressions of the proliferation marker Ki-67 and estrogen receptor in the uterus of mice with autoimmune premature ovarian failure (POF) induced by zona pellucida 3 peptide (pZP3).

METHODSAutoimmune POP models were established in 20 female BALB/c mice (7-8 weeks old) by immunization with pZP3 and another 20 mice served as the control group. The POP models were verified by vaginal cytology, serum sex hormones, ovary histomorphology and ZP3 antibody immunohistochemistry. The histomorphology and expressions of Ki-67, estrogen receptor α and estrogen receptor β in the uterus of the mice were detected.

RESULTSAutoimmune POP models were established successfully in 80% of the mice at 8 weeks after the immunization. Compared with those in the control group, the mice in the model group showed a smaller volume of the uterus, thinner endometrium and a reduced number of glands. The luminal epithelial cells, glandular epithelial cells and stromal cells in the uterus of the model mice all presented with a lower expression of Ki-67 than those in the control group, and Ki-67 translocation from the nuclei to the cytoplasm was found in the model group. The luminal epithelial cells, glandular epithelial cells and stromal cells showed positive ERα immunoreactivity in the model group but not in the control group. No obvious ERβ expression was found in the uterus in either of the groups.

CONCLUSIONpZP3 can induce autoimmune POP, cause suppressed proliferation of the endometrial epithelial cells and stromal cells, and reduce the cellular expression of ERα in the uterus of mice.

Animals ; Autoimmune Diseases ; metabolism ; Cell Nucleus ; Egg Proteins ; Endometrium ; Epithelial Cells ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Female ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Membrane Glycoproteins ; Mice ; Mice, Inbred BALB C ; Primary Ovarian Insufficiency ; metabolism ; Receptors, Cell Surface ; Stromal Cells ; Uterus ; metabolism ; Zona Pellucida Glycoproteins

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome alpha2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTCQ the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonicum.
Animals ; Antibodies, Helminth ; blood ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genes, Helminth ; Helminth Proteins ; genetics ; metabolism ; Immunization ; Liver ; parasitology ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Parasite Egg Count ; Proteasome Endopeptidase Complex ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Vaccines, Synthetic ; immunology

The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence(137 approximately 150) being completely conserved with huZP3(138 approximately 151) sequence) and specific huZP3-14 (amino acid sequence(327 approximately 340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a(22 approximately 176) and anti-r-huZP3b(177 approximately 348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Immune Sera ; immunology ; Immunization ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sperm-Ovum Interactions ; immunology ; Zona Pellucida Glycoproteins

AIMTo produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests.

METHODSThe human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed.

RESULTSRhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed.

CONCLUSIONRhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Acrosome Reaction ; drug effects ; Blotting, Western ; Cell Line ; Egg Proteins ; analysis ; genetics ; pharmacology ; Embryo, Mammalian ; Female ; Fluorescent Antibody Technique ; Gene Expression ; Glycoside Hydrolases ; metabolism ; Glycosylation ; Humans ; Kidney ; Male ; Membrane Glycoproteins ; analysis ; genetics ; pharmacology ; Receptors, Cell Surface ; analysis ; genetics ; Recombinant Proteins ; analysis ; pharmacology ; Zona Pellucida Glycoproteins