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1. chinaXiv:201711.02423 [pdf]

Expression of human il-15 in Pichia pastoris final V

Wei Sun; Yunxin Lai; Hongbo Li; Tao Nie; Ye Kuang; Xiaofeng Tang; Kuai Li; P. Rod Dunbar; Aimin Xua; Peng Li; Donghai Wu
Subjects: Biology >> Ecology

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.

submitted time 2017-11-17 Hits1212Downloads773 Comment 0

2. chinaXiv:201711.02430 [pdf]

CRISPRCas9-Mediated Deletionof Foxn1 in NODSCIDIL2rg?? Mice Results in Severe Immunodeficiency

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
Subjects: Biology >> Ecology

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.

submitted time 2017-11-17 Hits866Downloads473 Comment 0

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