Expression Of HIL-28A In Silkworm Bioreactor And Its Antitumor Evaluation

Sheppard has reported interlukin-28A (IL-28) in 2003, as one of novel interleukins, as known as interferonλ2 (IFN-λ2), which belongs to a new type of IFN-λs, also known as typeⅢinterferon. It contains the similar functions with typeⅠinterferon, including anti-virus, anti-proliferation immune regulation and other biological activities. The antiviral activity has been investigated both at home and abroad, using E. coli and mammalian cell expression systems usually. There are no relevant reports over the IL-28A expression based on Silkworm bioreactor and its impact on antiproliferation.The expression of exogenous proteins in Silkworm Bioreactor is an effective way to produce highly efficient biological products, which possesses many advantages, such as high expression level, stable expression product, better post processing, better security, easy to industrial production and the formation of our country's independent industry. Therefore, it is one of the most effective systems for the study on oral administration protein drugs. To further study anti-tumor effect of oral administration of recombinant human IL-28A (hIL-28A) in vivo and in vitro, and based on the non-transposon vector-mediated and the Bac-to-Bac system, respectively.we successfully constructed the recombinant expression vector which can express hIL-28A in BmN cells and silkworm pupae to investigate whether the oral administration of recombinant baculovirus expressing hIL-28A could improve the anti-tumor ability on nude mice. The following aspects are the detail research contents.1. The expression of hIL-28A in stable transformed BmN cell and the Anti-proliferation activity detectionThe recombinant expression vector pIZT/V5-His-hIL-28A was constructed to transfect the BmN cells, aiming to obtain the stable transformed BmN cell line which can continuously express hIL-28A gene, and investigate its anti-proliferation activity. After screening with Zeocin at a terminal concentration 300-400μg/mL for two months, we obtained the stable transformed BmN cells. The SDS-PAGE and Western blotting results illustrated that the transformed cells could express the recombinant hIL-28A protein of 26 kDa molecular weight. And ELISA result demonstrated the expression level of hIL-28A at 20.35 ng in 106 cells. Meanwhile, the anti-proliferation activity of hIL-28A was determined by MTT assay, the consequence showed that the 50% inhibitory concentrations (IC50) of the recombinant hIL-28A on A549 (Lung Cancer Cells), HL60 (Acute Promyelocytic Leukemia Cells), BEL-7402(Liver Cancer Cells) and M231 cells (Breast Cancer Cells) were approximately 3.21, 2.84, 6.29 and 9.32 ng/ml, respectively. In summary, hIL-28A can be stable expressed in the transformed BmN cell line, and the expressed recombinant hIL-28A harbors the anti-proliferation activity to tumor cells in vitro.2. The expression of hIL-28A gene in BmN cell and silkworm pupa using Bac-to-Bac systemThe hIL-28A was cloned into the downstream of polyhedron promoter of the pFastBacTMDual vector, and the recombinant virus Bacmid-EGFP-hIL-28A vector was finished using Silkworm/ Baculovirus expression vector system (Bac-to-Bac), to express the hIL-28A in BmN cells and silkworm pupae.The expression level of hIL-28A in BmN cell (harvested at 24 h to 144h post infection) was detected by ELISA. ELISA result showed that the amount of expressed hIL-28A protein in BmN cells at 96h post infected and blood lymph in silkworm pupae at 120h post infected reached highest level, 11.4μg/106 cells and 32.3μg/ml, respectively. The amount of expressed hIL-28A protein in silkworm pupae powder was 8.49mg/g. At the same time, BmN cells (96h post infection) and blood lymph of silkworm pupae (120h post infection) were detected by SDS-PAGE and western blotting. The results indicated that the molecular weight of expressed recombinant hIL-28A protein was about 26 kD.3. Effect of oral administration recombinant hIL-28A on anti-tumor ability in nude miceThe effects of hIL-28A contained silkworm pupae freeze-dried powder on the inhibitory of A549 (Lung Cancer Cells) and HL60 (Acute Promyelocytic Leukemia Cells) tumor cells in vivo were investigated by immunohistochemical methods. Compared with the control group, the size and weight of transplanted tumor in treated group have significantly decreased(p<0.05), and the ratio of spleen weight and body weight has increased significantly (p < 0.01).The expression level of PCNA(proliferating cell nuclear antigen) and VEGF(vascular endothelial growth factor) of transplanted tumor was decreased in treated group, compared with the control(p<0.05).The pathological changes were observed. In treated group, the number of cellular necrosis increased markedly compared with control group, and cell apoptosis can be observed. TUNEL (TdT-mediated dUTP-biotin nick end labeling) analysis showed that the number of apoptotic cells increased significantly in the treated group(p<0.05). These results illustrated that oral administration silkworm freeze-dried powder containing hIL-28A could inhibit tumor growth rate and size in nude mice, suggesting its anti-tumor effect may be related to reducing the level of positive expression of PCNA and VEGF in transplanted A549 and HL60 tumor cells, inducing apoptosis of tumor cells, as well as improving their own immunity in nude mice.The research results laid a foundation on the oral drug research and development of hIL-28A, and provided a new way for the pharmaceutical silkworm.