Eukaryotic Expression And Characterization Of Haemaphysalis Longicornis Transglutaminase

Haemaphysalis longicornis is a common blood-sucking ectoparasite on livestock,it sucks the blood of animals,causing damage to the host,and lead to many tick-borne diseases such as Babesia ovata,Theileria and so on.Transglutaminase(TGase)involved in formation of the arthropod hemolymph coagulation and skin stratum corneum,plays an important role in the immune process of arthropods.In this study,a full-length cDNA of transglutaminase was cloned from the cDNA library of H.Longicornis,and the gene of HlTGase was subcloned into E.coli,which was then induced to express the recombinant proteins rHITGase.However,both the yield and the enzyme activity of rHITGase was not satisfied.Considering H.longicornis belongs to an eukaryote,post-translational modification of eukaryotic proteins are often associated with its functions.An eukaryotic expression vector pPICZC/HITGase was constructed and el ectrotransformed into yeast Pichia pastoris.Analyzed with murine serum against rHITGase and antibody against polyhis by western blotting,the result showed that the recombinant HITGase(His-rHITGase)has been successfully expressed in the eukaryotic expression system.Induced in different conditions,the result showed that in pH 5.0 and 30℃ the optimal expression level is 250 μg/mL.According to the change in fluorescence intensity and wavelength when His-rHITGase catalytic biotinylated pentylamine covalent coupling to casein,showed that His-rHITGase was capable of catalyzing the crosslinking of pentyl amine and casein.At 25℃,when the incubation time up to 3 h,the catalytic activity can reach the maximum value.The crosslinking effect of casein catalyzed by His-rHITGase were observed by using SEM.Significant changes in the conformation of the proteins were found.Analysis on the biological activity of His-rHITGase and its enzymatic characteristics would help us further understand how it play physiological functions in H.longicornis,and provide a scientific basis for the prevention of H.longicornis.Whether transglutaminase from H.longicornis can be used as a food additive to improve the appearance and flavor in food and for the development of new biomedical materials remain to be studied.