分类： 生物学 >> 生态学 提交时间： 2017-11-17
Wei Sun
Yunxin Lai
Hongbo Li
Tao Nie
Ye Kuang
Xiaofeng Tang
Kuai Li
P. Rod Dunbar
Aimin Xua
Peng Li
Donghai Wu
摘要： Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the Nterminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75 mg/L rhIL-15 after 3 days of induction by methanol. The rhIL-15 was purified by Ni+-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from Pichia pastoris.
分类： 生物学 >> 生态学 提交时间： 2017-11-17
Xinru Wei
Yunxin Lai
Baiheng Li
Le Qin
Youdi Xu
Simiao Lin
Suna Wang
Qiting Wu
Qiubin Liang
Guohua Huang
Qiuhua Deng
Pentao Liu
Donghai Wu
Liangxue Lai
Yao Yao
Peng Li
摘要： Immunodeficient mice engrafted with either normal or cancerous human cells are widely used in basic and translational research. In particular, NOD/SCID/IL2rg−/− mice can support the growth of various types of human cancer cells. However, the hairs of these mice interfere with the observation and imaging of engrafted tissues. Therefore, novel hairless strains exhibiting comparable immunodeficiency would be beneficial. Recently, the CRISPR/Cas9 system has been used for efficient multiplexed genome editing. In the present study, we generated a novel strain of nude NOD/SCID/IL2rg−/− (NSIN) mice by knocking out Foxn1 using the CRISPR/Cas9 system from NOD/SCID/IL2rg−/− (NSI) mice. NSIN mice were deficient in B, T, and NK cells, which showed impaired T cell reconstitution and thymus regeneration after allogeneic bone marrow nucleated cell transplantation, and exhibited improved capacities of grafting both leukemic and solid tumor cells compared with NSI, NOG and NDG mice. Moreover, NSIN mice facilitated the monitoring and in vivo imaging of both leukemia and solid tumors. Therefore, our NSIN mice provide a new platform for xenograft mouse models in basic and translational research.
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