Prokaryotic expression of C-type lectin gene of Litopenaeus vannamei and immunologic activity evaluation of recombinant protein

Abstract

The studies on innate immune responses of Litopenaeus vannamei C-type lectin,were conducted by recombinant C-type lectin protein through expression in Escherichia coli.We used cell suspension culture to study the effect of C-type lectin on the cellular immune response of Litopenaeus vannamei.The experiment was divided into two groups:control group and recombinant protein(1.0 mg/mL).After 0,3,6,9,12,24 hours,we harvested the solutions and then assessed parameters respectively.The results showed that SDS-PAGE revealed the molecular weight of LvLec protein was 39 ku,which was consistent with the predicted size.Western-blotting revealed it could bind anti-His antibody,which proved that C-type lectin recombinant protein in Escherichia coli succeeded in expression.For the hemagglutination assay,massive agglutination could be observed after Escherichia coli was incubated with LvLec for 1 h.At the same time,LvLec protein had a significant effect on haemocyte count,cell viability and phagocytic activity of white shrimp Litopenaeus vannamei(P0.05).The extracelluar phenoloxidase activity of the LvLec protein group showed peak changes during 3-12 h,and at 9 h the extracelluar phenoloxidase activity was highest,then returned to the control level(P0.05).Therefore,crustacean C-type lectin not only had the function of hemagglutination,but also activated prophenoloxidase activating system,and probably played an important role in immune response.