Objective To isolate specific humanized anti-D-dimer scFv(single chain Fv) antibody from scFv phage libraries. Methods Isolate anti-D-dimer positive clones from Tomlinson I + J phage libraries by three rounds of panuing, then sequence monoclonal genes by bideoxy-mediated chain termination and express soluble scFv antibody; Pick out anti-D-dimer antibodies with high specificity and affinity by ELISA.Results After three rounds of selection from human scFv phage libraries Tomlinson I and J, 38 monclonal specific anti-D-dimer scFv fragments were selected. By polyclonal and monoclonal phage ELISA and gene sequencing, 20 different full-length monoclonal scFv phages were identified, the result of soluble scFv ELISA showed that 20 full-length monoclonal scFv were expressed smoothly. According to the result of soluble scFv ELISA, in 5 scFv antibodies with high value of A450 selected, 3 scFv antibody fragments showed high specific and affinity. Conclusion Antibody phage display was an effective, rapid method to isolate anti-D-dimer antibodies with high specificity and affinity.

BACKGROUNDIschemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165(VEGF165) gene in adeno-associated virus (AAV) gene delivery system.

METHODSHuman angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site HindIII, and the downstream of angiopoietin 1 contained restriction enzyme site BamHI. The upstream of VEGF165 contained restriction enzyme site BglII, and the downstream of VEGF165 contained restriction enzyme site BamHI. Using the multiple cloning sites (MCS) in plasmid pZero++ such as BamHI, BglII, HindIII, NotI, XhoI, XbaI, SalI, BspHI, KspI and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.

RESULTSDNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion, which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.

CONCLUSIONSuccessful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.

Angiopoietin-1 ; genetics ; Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Plasmids ; Vascular Endothelial Growth Factor A ; genetics

Objective To explore the relationship between recurrence rate and side effects of the treatment of varicose veins in lower extremity with lauromacrogol foam sclerosing agent.Methods The data of 62 patients(98 limbs)with varicose veins was collected,including 27 males (41 limbs)and 35 females (57 limbs).They were treated with 1％ lauromacrogol foam sclerosing agent combined with great saphenous vein high ligation and endovenous laser treatment.Statistical indicators included side effects such as pain,induration,hyperpigmentation,swelling,numbness and the recurrence rate.Results Two patients were lost,57 patients were injected once,further foam sclerotherapy was carried out again for 3 patients.Obvious abnormal varicose veins as well as the soreness and fatigue of lower extremity disappeared in all patients.Thirty-seven patients had pain and 43 patients developed superficial venous thrombotic sclerosis,among which 50 patients faded away after 1 to 3 months by taking diosmin.Thirty-one cases of pigmentation occurred and 20 disappeared after 1 to 3 months by applying the vitamin E whitening essence.Six patients with lower extremity ulcers recovered after 1 to 3 months.Through statistics analysis of the patients in 1-month follow-up,we found that the differences in recurrence rate after 6 months were statistically significant between group with pain induration reaction and group without (P ＜ 0.05).Among the patients without using the vitamin E whitening essence,there was a significant correlation between the duration of pigmentation and recurrence rate after 6 months.Conclusion In the process of foam sclerotherapy,the more pain induration reaction they had and the longer the duration of pigmentation sustained,the lower the recurrence rate would be accordingly.