Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.
Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/chemistry/*genetics/immunology/*isolation & purification ; Cloning, Molecular ; Dirofilaria immitis/chemistry/*genetics/immunology ; Disease Models, Animal ; Dogs ; Escherichia coli/genetics ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Tumor Markers, Biological/chemistry/*genetics/immunology/*isolation & purification

Human cytomegalovirus (HCMV) infection is an ubiquitous herpesvirus disease in human populations. It is rarely pathogenic to healthy adults, yet it may cause severe outcome to organ transplant recipients, the immunocompromised individuals and pregnant women. Using DNA from HCMV AD169 strain as template, the UL32 gene encoding pp150 protein fragment and the UL57 gene encoding MDBP protein fragment were amplified by PCR technique. After the construction of cloning vector pMD18-T-UL32, pMD18-T-UL57, pMD18-T-UL32/UL57 and expression vector pET-11a-UL32/UL57, the recombinant fusion proteins pp150/MDBP were induced with IPTG in BL21 host strain. The results showed that the relative molecular weight of recombinant fusion proteins pp150/MDBP is about 27 kD, the product of fusion proteins takes 17.45% in the total proteins in host bacteria, the analytical result was positive to the fusion proteins pp150/MDBP via Western blot technique, while the purified recombinant fusion proteins have strong antigenicity detected by ELISA and protein chip compared with whole virus antigens from HCMV. It was demonstrated that when used for the detection of serum from the clinical patients it has the same detection rate compared with the whole virus antigen. It needs further research for application.
Cytomegalovirus ; DNA-Binding Proteins ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; blood ; genetics ; immunology ; isolation & purification ; Phosphoproteins ; chemistry ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; blood ; genetics ; immunology ; isolation & purification ; Transcription Factors ; chemistry ; Viral Matrix Proteins ; chemistry

The use of tumor antigen specific antibody for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. In previous work, the human-mouse chimeric antibody fragment Fab' directed against CD20 was constructed from the new anti-CD20 antibody HI47 (a mouse IgG3, K). The chimeric antibody fragment Fab' could reduce its antigenicity, but the yield, quality and affinity of chimeric antibody fragment Fab' restrict its use. To improve affinity of chimeric antibody fragment Fab', a new phasmid pYZcpp3, which expresses chimeric antibody fragment F(ab')2, was constructed by adding a sequence encoding a small peptide, (CPP)3, to C-terminus of heavy chain constant region of chimeric antibody fragment Fab'. Using the pYZcpp3 to transform E. coli. 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment Fab' and F(ab')2 into periplasmic. The yield was up to 360 mg/L with the percent of F(ab')2 up to 45% in 19L fermentor by the high density fermentation technology. Without denaturation and renaturation, the F(ab')2 has possessed the native three-dimensional structure. The purity of F(ab')2 was more than 90% after the purification of protein G affinity chromatography and S200 size exclusion chromatography. The F(ab')2 could distinguish and bind to Raji cells (CD20+) by FACS. F(ab')2 could inhibit the proliferation of Raji cells in vitro by MTT, IC50 was 22.8 microg/mL. HI47 and its chimeric fragments F(ab')2 induced a significant level of apoptosis (23.5%, 20.8%, respectively), independent of any cross-linking agents, in Raji cells after 24 h incubation. The chimeric antibody fragment F(ab')2 directed against CD20 is possible to apply to tumor therapy in clinic in the future.
Antigens, CD20 ; immunology ; Apoptosis ; Escherichia coli ; genetics ; Fermentation ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; isolation & purification ; therapeutic use ; Lymphoma, B-Cell ; therapy ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; therapeutic use

OBJECTIVETo clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.

METHODSBased on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.

RESULTS AND CONCLUSIONThe migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; Base Sequence ; Blotting, Western ; Cell Adhesion Molecules ; genetics ; immunology ; Cell Line, Tumor ; Cloning, Molecular ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeletal Proteins ; genetics ; immunology ; DNA, Complementary ; chemistry ; genetics ; Escherichia coli ; genetics ; Humans ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo study the role of PAK6 in prostate cancer by cloning PAK6-N terminal sequence into E.coli and preparing its polyclonal rabbit antibody to detect PAK6 expression in prostate cancer.

METHODSBased on human PAK6 cDNA sequence, we designed a pair of primers to amplify the PAK6-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 via EcoRI/XhoI sites, and the recombinant plasmids were identified by enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL21 cells, the GST-PAK6-N fusion protein was expressed with IPTG induction. Glutathione-Sepharose beads were used to purify GST- PAK6-N fusion protein. Anti-PAK6 polyclonal antibody was produced by immunizing rabbits with purified GST-PAK6 N-terminal fusion protein. Anti-PAK6 polyclonal antibody was purified by protein A beads and used for detection of PAK6 expression in 3 prostate cancer specimens.

RESULTS AND CONCLUSIONWe cloned PAK6-N terminal gene fragment successfully, purified GST-PAK6 N-terminal fusion protein, and obtained polyclonal rabbit PAK6 antibody. Immunohistochemistry indicated that PAK6 expressed in the stroma instead of the cancer cells in prostate cancer. All of the 3 prostate cancer specimens showed positive staining in the stroma, suggesting that PAK6 may participate in the stroma-cancer cell interaction in prostate cancer.

Aged ; Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Male ; Polymerase Chain Reaction ; Prostatic Neoplasms ; enzymology ; genetics ; Rabbits ; Recombinant Fusion Proteins ; immunology ; metabolism ; Sequence Analysis, DNA ; p21-Activated Kinases ; genetics ; immunology ; metabolism