| Human interferon-γ (hIFN-γ) is a common natural glycoprotein, a member ofinterferon (Interferon, IFN) family, possessing a variety of biological activities.hIFN-γ can achieve the anti-viral effect mainly by acting on macrophages and T cellsto regulate immune. In vitro studies have found that IFN-γ can inhibit the proliferationof cancer cells in vitro and induce apoptosis. IFN-γ also can be effective for a varietyof xenograft model, indicating a better anti-tumor potential. The incidence andmortality rates of breast cancer are high and it is the most common female cancer withpoor prognosis. The abnormal biological behaviors are key factors leading todifficulty to cure for breast cancer. The above indicates hIFN-γ may become a strategyfor breast cancer treatment, may reduce the recurrence and prolong survival ofpatients with breast cancer. Large quantity high-purity hIFN-γ is important for clinicalresearch and their mechanisms. Due to the limitation of acquiring natural hIFN-γ, itrequires the use of bio-engineering technology for large-scale preparation. Theprevious expression systems are improper for their complex process operation, highproduction costs and lower yields. If the efficient bio-engineering vector can beemployed for obtaining rhIFN-γ with the similar functions as natural hIFN-γ, thesource problem of hIFN-γ can be well solved.In this study, the Pichia pastoris expression system was employed to producerhIFN-γ with similar components as hIFN-γ. Meanwhile, the biological activity ofrhIFN-γ was evaluated by in vivo and in vitro studies,including the following aspects:1Preparation and optimized fermentation conditions of rhIFN-γThe sequence of hIFN-γ was designed according to GeneBank (accessionnumber: BC070256).After the gene synthesis, hIFN-γ was connected to pPICZvector and then transferred to construct engineered bacteria. The successful-expreesedengineered bacteria were hereby selected. The optimal condition for Pichia pastoris toexpress rhIFN-γ was pH of6.0at the temperature of28℃.And the optimal inductiontime point was the6thday.A Total amount of0.601g purified rhIFN-γ was obtained from5L medium. The Tricine-SDS-PAGE electrophoresis and high performanceliquid chromatography showed a purity of95.6%for rhIFN-γ.2Studies of in vitro inhibitory effect of rhIFN-γThe human breast cancer MDA-MB-231cells were treated with rhIFN-γ at thedose of0,100,500and1000U/mL, respectively. MTT shows the rhIFN-γ can inhibitcell proliferation of MDA-MB-231cells at24th,48thand96thh after treatment. TheHoechst staining and flow cytometry indicate that rhIFN-γ can increase the apoptoticindex and apoptosis rate in a dose-dependent manner. The DHE staining showsrhIFN-γ can reduce the ROS levels in a dose-dependent manner. The western blotshow rhIFN-γ can decrease levels of Bcl-2and p-Akt/Akt, and increase levels ofCleaved caspase-3and Bax in a dose-dependent manner.3Studies of in vivo inhibitory effect of rhIFN-γThe breast cancer xenografts in nude mice model were treated with rhIFN-γ atthe dose of0,100,500and1000U/g, respectively. The rhIFN-γ can decerase the tumorsize in a dose-dependent manner. The immunohistochemical staining shows thatrhIFN-γ can reduce the expression of MMP-2and MMP-9in the tumor tissue in adose-dependent manner. Elisa shows rhIFN-γ can reduce the VEGF levels of tumortissue and serum in a dose-dependent manner. The western blot shows rhIFN-γ candecrease the level of p-Akt/Akt in a dose-dependent manner.In conclusion, Pichia pastoris stably secret,and express rhIFN-γ and the newmethod for large-scale purification of the recombinant protein was established. The invivo and in vitro studies proved rhIFN-γ can inhibit the development of breast cancer. |
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