OBJECTIVETo develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.

METHODSA previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.

RESULTSAn about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.

CONCLUSIONSThe single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; DNA-Binding Proteins ; HeLa Cells ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Molecular Sequence Data ; Sequence Analysis, DNA ; Telomerase ; immunology

OBJECTIVE: To evaluate the detection of humen-lung-specific X protein (LUNX) gene in micrometastases of patients with non-small cell lung cancer. METHODS: The expression of LUNX gene in tumor tissue, lung and lymph nodes was detected by reverse transcriptase-polymerase chain reaction(RT-PCR) both in 43 non-small-cell lung cancer patients (the experimental group) and 15 lung benign patients (the control group). LUNX mRNA expression in clinic pathology,stage of cancer cell differentiation, clinic stage, age, sex, smoking history, and 4 lung cancer blood markers (CEA,CA125,NSE, and CYFRA211) were evaluated. RESULTS: The expression of LUNX gene was positive in the 2 groups. LUNX gene expression was positive in 33 of the 87 lymph nodes of the 43 patients in the experimental group (37.93%), and in 2 of the 26 lymph nodes in the control group (7.69%). The LUNX mRNA positive in the lymph nodes was closely related to the pathological type, cancer cell differentiation and clinic stage(r=0.660,0.500,0.460; P=0.011,0.017,0.022, all P<0.05), while not closely related to age, sex, smoking history and 4 lung cancer blood markers (CEA,CA125, NSE, and CYFRA211) (r=0.111, 0.135,0.083,0.354; P=0.739,0.714,0.773,0.125,all P>0.05). CONCLUSION The LUNX mRNA expression detected by RT-PCR is more sensitive than by traditional ways. The expression of LUNX gene mRNA in the lymph nodes is a valuable index for the detection of micrometastases in patients with non-small cell lung cancer.
Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Female ; Glycoproteins ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Lymph Nodes ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Phosphoproteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Objective To compare vein valve function following pharmacomechanical thrombolysis (PMT) with simple catheter-directed thrombolysis (CDT) for deep vein thrombosis. Methods We retrospectively analyzed the clinical data of sixty patients who suffered acute lower extremity deep vein thrombsis in our hospital between October 2016 and March 2017. All patients underwent contralateral preprocedural duplex and bilateral postprocedure duplex to access patency and valve function. The patients were divided into three groups including a group A with catheter-directed thrombolysis (CDT) alone (36 patients with 20 males and 16 females at average age of 56 years), a group B with PMT alone (15 patients with 8 males and 7 females at average age of 55 years), and a group C with PMT combined CDT (9 patients with 4 males and 5 females at average age of 56 years). The valve function was compared among the Group A, Group B and Group C. Results There were 40.0% (24/60) patients with bilateral femoral vein valve reflux, 40.0% (24/60) patients with unilateral femoral vein valve reflux (all in the treated limbs), 20% (12/60) patients had no reflux in both limbs. Of the limbs treated with CDT alone, PMT alone and PMT combined CDT, the rate of valve reflux was 38.9% (14/36), 33.3% (5/15), and 55.6% (5/9) respectively (P=0.077). Conclusion In the patients suffering acute DVT, PMT or PMT combined CDT does not hamper valve function compared with CDT alone.