Application of a single-chain fragment variable (scFv) antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

Background: Hydatid disease is a serious parasitic disease threatening public health. Because of its rarity in non-endemic coastal areas, determining the nature and origin of a chronic, enlarged liver cystic mass is challenging in these regions. Under these circumstances, physicians need a confirmatory diagnostic tool beyond immunological and radiological examinations. This study investigated a novel human single-chain fragment variable (scFv) antibody for the confirmative diagnosis of 18 atypical hydatid disease cases in non-endemic coastal areas. Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P b 0.05). The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

Construction of a mutant human proinsulin gene with signal peptide code and detection of its expression in COS-7 cells / 重庆医科大学学报

Objective:To construct a mutant of human proinsulin with signal peptide code,so that it may be expressed in eukaryotic system to produce mature human insulin in COS-7 cells.Methods:Human proinsulin gene was mutated by the polymerase chain reaction (PCR) for the site-directed mutagenesis method.Two furin recognized sites of Arg-X-Lys-Arg-motifs were introduced into human proinsulin.Signal peptide,Kozak sequence and restriction enzyme cutting site were added and cloned into pcDNA3.1(+) vector and verified by sequencing analysis.Eukaryotic expression vector was constructed and transfected into COS-7 cells.Insulin were evaluated respectively with RT-PCR,radioimmunoassay and Western blot analysis.Results:The sequencing analysis showed that the mutation sites were correct,and the mutant of human proinsulin was constructed successfully.Human insulin mRNA was highly expressed in transfected COS-7 cells,and secretive recombinant human insulin was detected in the culture supernatant of transfected COS-7 cells by Western blotting.Conclusion:A mutant human proinsulin eukaryotic expression vector is obtained and secretive recombinant human insulin is successfully expressed in COS-7 cells.

A kind of new albumin microbubble enhances EGFP gene expression in Cos-7 cell by ultrasound-mediated microbubble destruction / 中华超声影像学杂志

ObjectiveTo find a new approach of transfecting the objective gene safely and effectively according to the cavitation effect of ultrasound mediated microbubble destruction which could increase the permeability of macromolecule (such as DNA) across the eucell membrane.MethodsEGFP gene was transfected to Cos 7 cell as mark one in vitro by ultrasound mediated microbubble destruction and liposome as control. The transfection effect was surveyed by laser microscope and flow cytometry qualitively and quantitively. Trypan blue staining was adopted to measure the cell vitality.ResultsUltrasound mediated microbubble destruction at 0.8 MHz , 1.0 W/cm 2, 10% duty cycle, 60 s can get the most stable effective expression of EGFP gene in Cos 7 cell and no cytotoxicity. ConclusionsAlbumin microbubble made by us is a new and effective carrier of objective gene in gene therapy. At some specific ultrasound condition, microbubble destruction can enhance the objective gene transfection and expression and have a good targetivation. Ultrasound mediated destruction of albumin coated microbubble is a promising method in gene therapy.