Construction and application of EV71 ScFv high-throughput cassette expression system / 中华实验和临床病毒学杂志

Objective@#To construct a method to express ScFv antibody from PCR products, and use it in phage display for high-throughput ScFv expression.@*Methods@#Cytomegalovirus (CMV) promotor, ScFv and BGH-Poly A gene fragments were amplified by PCR. Overlapping PCR was used to form a tandemly linear cassette gene. By transiently transfected into 293T cells, ScFv antibodies expression of cassette gene were tested by Western blot, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA). Ninety-six clones of antibody genes in phage library were selected and expressed by cassette expression system. The expression level was evaluated and analyzed.@*Results@#Three fragments were obtained and a cassette expression system formed. Cassette expression system worked successfully in 293T cells, as a Mr.38×103 brand could observed in Western blot assay. The expressed antibody could specifically bind to its antigen by result of ELISA and IFA. This cassette expression system could also be used in phage display for high-throughput panning.@*Conclusions@#The cassette expression system was constructed successfully and high-throughput antibody expression has been achieved.

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.

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Construction and protective effect of targeting antimicrobial peptide against Toxoplasma / 中国血吸虫病防治杂志

Objective To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma fused to magainin, and observe its protective effect against Toxoplasma in infected mice. Methods The S1 scFv antibody gene amplified from phagmid S1/pIT-2 fused to magainin was cloned into procaryotic expression vector pET-32c. The recombinant plasmid S1M/pET-32c proved by DNA sequencing was transformed into E.coli BL21, and induced for fusion expression of S1M with IPTG. The expressed S1M was purified with Ni 2+ chelating HiTrap HP column and detected with SDS-PAGE. The effect of reduction of infection of Toxoplasma was observed through in vivo and in vitro experiments in mice. Results The fused gene of S1 and magainin was successfully cloned into procaryotic expression vector pET-32c proved by DNA sequencing. The recombinant S1M protein about 43 kDa was expressed in E.coli as inclusion body, and prepared with Ni 2+ column purification. Tachyzoite of Toxoplasma preincubated with S1M showed decreased infectivity in mice, the result of in vivo experiments showed that mice treated with S1M hadlonger survival time than the mice untreated. Conclusion The purified targeting antimicrobial peptide S1M could reduce the infectivity of tachyzoites of Toxoplasma in a certain extent and has a potential value for biological therapy of toxoplasmosis; otherwise, the constructed targeting antimic robial peptide S1M also provides a new model for biological therapy of toxoplasmosis.