Eukaryotic Expression Of Porcine CD40L And Its Immunological Activity In Vitro

Objective:Express porcine CD40L by constructed Pichia pastoris strain. Assess its immunological activity in vitro.Methods:Porcine CD40L was expressed by Pichia pastoris yeast and subsequent protein purification was performed by Ni-Sepharose 6 Fast flow column and Poros 50 HQ Strong Anion Exchange column. Protein was analyzed by SDS-PAGE and Western Blot. Concentration was measured by BCA Protein Kit. Purified protein was marked with Alexa Fluor(?) 488. The obtained porcine pulmonary alveolar macrophages were distinguished if they were CD40 positive or not by cells Indirect Immunofluorescence and Western Blot. The binding of marked protein and CD40+ PAM was observed by fluorescence microscope.The safety concentration of pCD40L to PAM was detected by MTT method. Low dose(0.4?g/mL), middle dose(4?g/mL) and high dose(40?g/mL) groups were set up upon protein concentration gradient, as well as PBS group and blank group as control. After 12h and 24h, total RNA was extracted from cells of each group using Trizol method, and the changes of IL-1? and TNF-? in the level of gene expression were detected compared with reference genes GAPDH. The protein level changes of this two kind of cytokines were detected by Elisa using the cell culture supernatant.All the data was analysed by GraphPad Prime5.Results: ?We got pCD40L protein of high purity, and its concentration was 0.8mg/mL after expression and purification.?Porcine pulmonary alveolar macrophages were CD40 positive cells.? pCD40L marked with fluorescence was observed by fluorescence microscope that they can bind to CD40+ PAM.?The safety concentration of pCD40L to PAM was less than or equal to 40?g/mL at 24h.?The results of relative real time PCR showed that, compared with PBS group and blank control group, the mRNA expressions of IL-1? and TNF-? of pCD40L group increased significantly. They were influenced by different time points and concentrations, respectively, high dose(40?g/mL) group were P<0.001.?The results of Elisa showed that, compared with PBS group and blank group, the protein level of IL-1? and TNF-? of high dose(40?g/mL) group were increased significantly (P< 0.001) at 12h.The protein level of IL-1? of middle dose(4?g/mL) group were increased significantly(P<0.01) compared with the blank group. There was no difference of both the IL-1? and TNF-? levels between low dose(0.4?g/mL) group and control groups. At 24h, the IL-1? levels of high dose(40?g/mL) and middle dose(4?g/mL) groups were increased significantly(P<0.01) compared with the PBS and blank control groups, while there was no difference of low(0.4?g/mL) group. The TNF-? levels of all protein groups and PBS group were increased significantly(P< 0.001) compared with the blank groups, but there was no significant difference between protein groups and PBS group.Conclusion:Porcine CD40L were obtained successfully. It was approved that pCD40L can bind to PAM which were CD40 positive cells. pCD40L can increase the level of proinflammatory factors of IL-1? and TNF-? could be increased by incubation with pCD40L at the genetic level and protein level.This protein had biological activity and immunological activity in vitro.