Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
Acrosome ; immunology ; Animals ; Antibodies ; analysis ; Blotting, Western ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; immunology ; Escherichia coli ; Humans ; Immunohistochemistry ; Male ; Muramidase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; Semen ; immunology ; Spermatozoa ; immunology ; Testis ; immunology

To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
Animals ; Antibodies, Helminth ; blood ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Female ; Genes, Helminth ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccination

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a(+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control
Animals ; Antibodies, Helminth ; blood ; Blotting, Western ; Cloning, Molecular ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Genetic Vectors ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Schistosoma japonicum ; genetics ; metabolism ; Schistosomiasis japonica ; prevention & control ; Vaccines ; immunology

This study was purposed to prepare eukaryotic expression vector of recombinant human platelet CD36 gene. The total RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region (Gly30-Asn439) was amplified by RT-PCR. The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E. coli DH5α. The positive recombinant pMD18-CD36 plasmid was screened. After sequencing, this combinant vector was inserted into the transient eukaryotic expression vector pTE2, the pTE2-s-CD36-10 His transient eukaryotic expression vector was constructed. The recombinant CD36 Gly30-Asn439 expressed by HEK-293 cells was purified with Ni(2+) 2NTA chromatography. The results showed that 1.4 kb cDNA was amplified by RT-PCR, sequencing of the cDNA indicated the sequence was exactly the same to that in Genbank NM_001001547.2. The HEK293 cells with the plasmid were transfected, and SDS-PAGE confirmed that the transfect HEK293 cells expressed the human CD36 antigen extracellular protein fragments. Western-blot showed that the monoclonal antibody could recognize the recombinant CD36 with the sensitivity of 8 ng. It is concluded that the CD36 Gly30-Asn439 can be highly expressed by human embryonic kidney cells (HEK293), and the monoclonal antibody with biological activity has been obtained, which provide the basis for further study on platelet transfusion refractoriness.
Animals ; Antibodies, Monoclonal ; genetics ; isolation & purification ; Blood Platelets ; immunology ; Blotting, Western ; CD36 Antigens ; genetics ; immunology ; DNA, Complementary ; Genetic Vectors ; Humans ; Mice ; Plasmids ; Platelet Transfusion ; Reverse Transcriptase Polymerase Chain Reaction

Treatment with interferon beta (IFN-beta) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-beta are associated with a loss of IFN-beta bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-beta in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-beta medications by ELISA. The NAbs against IFN-beta were measured in BAb-positive MS patients receiving IFN-beta using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-beta treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-beta. In conclusion, the three IFN-beta preparations have different degrees of immunogenicity.
Adolescent ; Adult ; Antibodies/*blood/immunology ; Antibodies, Neutralizing/*blood/immunology ; Cross Reactions ; DNA, Complementary/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interferon-beta/*immunology/therapeutic use ; Male ; Middle Aged ; Multiple Sclerosis/drug therapy/*immunology ; Myxovirus Resistance Proteins/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.
Animals ; Antibodies, Viral/blood ; Base Sequence ; Cell Line ; Classical Swine Fever/immunology/*virology ; Classical swine fever virus/*genetics/immunology/pathogenicity ; Cloning, Molecular ; DNA, Complementary/genetics/immunology ; Immunization/methods/standards/veterinary ; Molecular Sequence Data ; Neutralization Tests/veterinary ; RNA, Viral/chemistry/genetics ; Recombinant Proteins/immunology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Analysis, DNA ; Specific Pathogen-Free Organisms ; Swine ; Virulence

Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.
Calibration ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genome, Viral ; genetics ; Infectious Anemia Virus, Equine ; immunology ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; Sequence Analysis ; methods ; Vaccines, Attenuated ; genetics ; Viral Envelope Proteins ; genetics ; Viral Vaccines ; genetics

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing β1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.
Amino Acid Sequence ; Blotting, Western ; Carbohydrate Sequence ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; Down-Regulation ; Epitopes ; genetics ; immunology ; Fucose ; metabolism ; Fucosyltransferases ; chemistry ; deficiency ; genetics ; immunology ; Glycoproteins ; chemistry ; genetics ; immunology ; Molecular Sequence Data ; Pentosyltransferases ; chemistry ; deficiency ; genetics ; immunology ; Polysaccharides ; chemistry ; immunology ; Protein Engineering ; methods ; RNA Interference ; Species Specificity ; Tobacco ; cytology ; genetics ; Xylose ; metabolism

Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.
Algal Proteins ; Antibodies ; metabolism ; Carboxypeptidases ; biosynthesis ; chemistry ; genetics ; immunology ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Hydrolysis ; Proprotein Convertases ; biosynthesis ; chemistry ; genetics ; immunology ; RNA, Plant ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Spinacia oleracea ; enzymology ; genetics