A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis

Abstract

Amaranthaceae pollen allergy incidence has increased in the last years due to the desertification process occurring in many countries. In some regions of Spain,Salsola kali is the main cause of pollinosis at almost the same level than olive and grass pollen.Sal k 1 -the sensitization marker of S. kali pollinosis- is actually used in clinical diagnosis, but it is purified at a low yield from pollen. Thus, the aim of the study consists of the production of a recombinant isoform of Sal k 1 able to span the structural and immunological properties of the natural isoforms present in S. kali pollen and validate its potential use for diagnosis. Methods: Sal k 1-encoding cDNA was amplified by PCR, cloned in pET41b vector and used to transform BL21(DE3) E. coli cells to produce the recombinant allergen. Immunoblotting, ELISA, basophil activation and skin prick test were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen sensitized patients and anti-Sal k 1 monoclonal and polyclonal antisera were used. Results: rSal k 1 was produced in E. coli with a final yield of 7.5 mg/L of cell culture. The expressed protein was isolated,purified to homogeneity, and structurally and immunologically validated against the natural form –nSal k 1- isolated from pollen as a useful diagnostic tool. In addition, Sal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with related and non-related pollen sources such as P.acerifolia and Oleaceae members. Conclusions: rSal k 1 expressed in bacteria maintains its structural and immunological properties intact in comparison to nSal k 1.rSal k 1 spans the immunological properties of most of the natural isoforms found in pollen, and thus, might substitute nSal k 1 in clinical diagnosis.