Expression Of Chicken Interleukin-18 Maturation Protein Gene In Insect Cells And Identification Of Bioactivity Of Its Expressed Protein

Interleukin-18(IL-18), a novel discovered cytokine, which is the potent inducor of IFN-γproduction, was firstly cloned by Okamura from Hepar in 1995 and mainly secreted from cells of monocyte macrophage system. It has been demonstrated that IL-18 plays a major role in immunoregulation and protection, and has large potential in host defense against various micro- organic infection and tumor.These research on human beings and animals were identified by many documents recently. However, the study on chicken interleukin-18(ChIL-18) lagged off the mammal interleukin-18. Previously, the gene of ChIL-18 was cloned, but further study is not performed.The discovery of ChIL-18 gene is very meaningful for both the study on comparative immuneology and the therapeutics for poultry diseases.In 2001, the high expression of the mature chicken interleukin- 18(mChIL-18) in E.coli had been finished in our laboratory. But the protein lost natural structure because of the fault of the system, such as noglycosy- lation form.So people have some questions about the bioactivity of the protein expressed by E.coli. In the present study, how to obtain the efficiently expressed mChIL-18 protein with biological activity was studied.In the experiment, the insect/baculovirus system was selected to express the chIL-18 mature protein and the biologic activity was detected. The mature chicken interleukin-18(mChIL-18) gene was amplified by designing one pair of primers according to the sequence which have been published.Recombinant baculovirus transfer vector was constructed by inserting cDNA copy of mChIL-18 gene into the downstreams of PH promoter of the baculovirus expression plasmid pFastBacTM HTb. Finally, the constructing eukaryotic expression plasmid named as pFastBacTM HTb-mChIL-18. After constructing pFastBacTMHTb-mChIL-18 plasmid, we make it transformate into DH10BacTM competent cells. And a recombinant bacmid(Bacmid-ChIL18) was obtained by there times blue/white spot selection and PCR identification by M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα- complementation region to facilitate PCR analysis). In order to harvest the P1 recombinant aculovirus, we transfected the purified bacmid into Spodoptera frugiperda 9(sf9) by using a cationic lipid such as Cellfectin Reagent. Raised the virus titre by infecting sf9 many times and infected the sf9 with the higher baculoviral stock(107~108 pfu/mL) for expressing protein and harvested the supernatant and cells in different times. The expressed mChIL-18 protein was analyzed by SDS– PAGE, detected by Western blotting and IFA, and the results demonstrated that recombinant protein of 23kDa in molecular mass was expressed successfully in insect cells.The biological activities of purified fusion protein, including of the proliferation of lymphocyte, inducing IFN-γproduction and inhibiting the Vesicular stomatitis virus(VSV) activity, were detected.The experiment of MTT showed that every concentration of mchIL-18 fusion protein could enhance proliferation of lymphocyte, and the effect was best when it was 200ng/mL and the proliferation exponent was 2.87. The experiment of VSV inhibition indicated that the mchIL-18 fusion protein could induce spleenocytes to produce IFN-γwith high activity when it was more than 100ng/mL, and the amount of mChIL-18 fusion protein and its inducing effect on IFN-γhad a direct proportion. In the experiment of VSV inhibition, different dilution of IFN-γwas used and it could protect the cells when it was more than 10U/mL. The result showed that mChIL-18 fusion protein could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. So it can be concluded that mChIL-18 was expressed in sf9 and the expression product has functional activities.