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| Expression of recombinant canine interleukin-2 in Pichia pastoris and identification of its bioactivity |
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Abstract To obtain recombinant canine interleukin-2(cIL-2) with natural bioactivity expressed in Pichia pastoris, the cDNA of canine IL-2 was amplified by reverse transcription polymerase chain reaction(RT-PCR) from the total mRNA of the lymphocyte from canine blood, which stimulated with ConA for 20h. The amplified fragment was cloned into pMD18-T vector and confirmed it was identical to that published in GenBank. The fragment was inserted into expression vector pPICZα-A. After linearized by SacⅠ, the recombinant plasmid was transformed into X-33 strain. The recombinant strain was isolated and identified by PCR. After induced by methanol, the recombinant protein was examined by SDS-PAGE. The result of SDS-PAGE in concentrated fermentation supernant showed that the molecular weight of the recombinant protein was approximately 20kDa. The expressed protein was larger,which may be due to glycosylation of protein. The result of MTT assay proved the recombinant protein could induce canine T lymphocytes proliferation in vitro. It concluded that canine recombinant IL-2 expressed in Pichia pastoris had good bioactivity.
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Received: 28 November 2006
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SHEN Xue-Jing, ZHANG Er-Qin, XU Qian-Ru, LIU Lin-Ke, WAN Bo, LIU Yun-Chao, SUN Ya-Ning, ZHAO Dong, NIU Xiang-Xiang, DENG Rui-Guang, ZHANG Gai-Ping. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability[J]. 农业生物技术学报, 2019, 27(2): 204-211. |
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