[Screening of antagonist peptide of BLyS from C7C phage display peptide library].

AIM: To screen an antagonist peptide of BLyS from C7C phage display peptide library. METHODS: C7C phage display peptide library was screened with BLyS. Indirect ELISA, competitive ELISA and MTT colorimetry were used to identify positive phage clones. RESULTS: After 3 rounds of screening, the gradual increase of the ratio of output to input and specific enrichment had been achieved. Two phage clones that could inhibit the BLyS activity were identified. CONCLUSION: Two phage display antagonist peptides of BLyS were obtained.

Imaging diagnosis of 12 patients with hepatic tuberculosis.

AIM: To assess CT, MR manifestations and their diagnostic value in hepatic tuberculosis. METHODS: CT findings in 12 cases and MR findings in 4 cases of hepatic tuberculosis proved by surgery or biopsy were retrospectively analyzed. RESULTS: (1) CT findings: One case of serohepatic type of hepatic tuberculosis had multiple-nodular lesions in the subcapsule of liver. Parenchymal type was found in 10 cases, including multiple, miliary, micronodular and low-density lesions with miliary calcifications in 2 cases; singular, low-density mass with multiple flecked calcifications in 3 cases; multiple cystic lesions in 1 case; multiple micronodular and low-density lesions fusing into multiloculated cystic mass or "cluster" sign in 3 cases; and singular, macronodular and low-density lesion with multiple miliary calcifications in 1 case. One case of tuberculous cholangitis showed marked dilated intrahepatic ducts with multiple flecked calcifications in the porta hepatis. (2) MR findings in 4 cases were hypointense on both T1-weighted imagings and T2-weighted imagings in one case, hypointense on T1-weighted imagings and hyperintense on T2-weighted imagings in 3 cases. Enhanced MR in 3 cases was slightly shown peripheral enhancement or with multilocular enhancement. CONCLUSION: Various types of hepatic tuberculosis have different imaging findings, and typical CT and MR findings can suggest the diagnosis.

[The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis].

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.

[Preparation of phage-displayed single chain variable fragments of monoclonal antibody MC5 recognizing colorectal carcinoma].

BACKGROUND & OBJECTIVE: MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study. The aim of this study was to prepare single chain variable fragments (ScFv) of MC5. METHODS: mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs. After two rounds of panning with cell line SW480 highly expressing MC5-binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA. RESULTS: The VH, VL, and ScFv DNAs were about 340 bp, 320 bp, and 750 bp, respectively. Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen. CONCLUSION: The phage-displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3.

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.