1.Stable expression of mouse IFN-λ2 in CHO cells and its biological activity analysis
Yulan YAN ; Lixue YUAN ; Yang LIU ; Wenyan CAO ; Xuefeng BU ; Zhigao BU ; Jinxu ZHENG
Chinese Journal of Microbiology and Immunology 2010;30(2):104-109
Objective To express mouse IFN-λ2 stably and study its biological activity. Methods Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by ve-sicular stomatitis virus(VSV) and then subcloned to eukaryotic expressing vector PCAGG-EGFP. The recom-binant was transfected into CHO cells. VSV * GFP-B16 system was used to measure the antivirus activity. The constructed cell line MDBK-Mxp-Luc was used to study the character of Mx1 protein induced by the mIFN-λ2. Results The recombinant pMD18-T-mIFN-λ2 was digested by two kinds of enzyme, Sac I and Xho I, to produce the fragment was of 582 bp, and of which the sequence analysis of sequence shows it was entirely consistent with the nucleotide sequences reported in GenBank. PCAGG-EGFP-mIFN-λ2 eukaryotic expressing vector was constructed successfully and expressed stably in CHO cells, and the mRNA of mIFN-λ2 was verified expressing in CHO-PCAGG-EGFP-mIFN-λ2 cell line by RT-PCR. The antivirus activity of in the supernatant secreted by the CHO-PCAGG-EGFP-mIFN-λ2 cell line was 10~4 AU/ml. The mIFN-λ2 pro-tein can could induce the expression of the antivirus protein Mx1, and the expression of Mx1 protein induced by mIFN-λ2 enhanced with time going, 9 to 12 hours achieved the peak, 24 hours vanished. Conclusion Gene cloning of mIFN-λ2 was successful. The eukaryotic expressing vector of mIFN-λ2 was constructed suc-cessfully and expressed stably in CHO cells, and its product has obvious antivirus activity in vitro. And the antivirus activity of the product was closely correlated with inducing expression of antivirus protein Mx1.
2.Small interfering RNA delivery to the neurons near the amyloid plaques for improved treatment of Alzheimer׳s disease.
Qian GUO ; Xiaoyao ZHENG ; Peng YANG ; Xiaoying PANG ; Kang QIAN ; Pengzhen WANG ; Shuting XU ; Dongyu SHENG ; Liuchang WANG ; Jinxu CAO ; Wei LU ; Qizhi ZHANG ; Xinguo JIANG
Acta Pharmaceutica Sinica B 2019;9(3):590-603
Gene therapy represents a promising treatment for the Alzheimer׳s disease (AD). However, gene delivery specific to brain lesions through systemic administration remains big challenge. In our previous work, we have developed an siRNA nanocomplex able to be specifically delivered to the amyloid plaques through surface modification with both CGN peptide for the blood-brain barrier (BBB) penetration and QSH peptide for -amyloid binding. But, whether the as-designed nanocomplex could indeed improve the gene accumulation in the impaired neuron cells and ameliorate AD-associated symptoms remains further study. Herein, we prepared the nanocomplexes with an siRNA against -site amyloid precursor protein-cleaving enzyme 1 (BACE1), the rate-limiting enzyme of A production, as the therapeutic siRNA of AD. The nanocomplexes exhibited high distribution in the A deposits-enriched hippocampus, especially in the neurons near the amyloid plaques after intravenous administration. In APP/PS1 transgenic mice, the nanocomplexes down-regulated BACE1 in both mRNA and protein levels, as well as A and amyloid plaques to the level of wild-type mice. Moreover, the nanocomplexes significantly increased the level of synaptophysin and rescued memory loss of the AD transgenic mice without hematological or histological toxicity. Taken together, this work presented direct evidences that the design of precise gene delivery to the AD lesions markedly improves the therapeutic outcome.