鼠IFN—λ2 CHO细胞系建立及生物学活性的研究  

Stable expression of mouse IFN-λ2 in CHO cells and its biological activity analysis

作　　者：严玉兰[1,2] 袁利学[1] 刘洋[1] 曹文雁[3] 步雪峰[1] 步志高[3] 郑金旭[2] 

机构地区：[1]江苏大学附属人民医院呼吸科,镇江212002 [2]江苏大学基础医学与医学技术学院,镇江212013 [3]中国农业科学院哈尔滨兽医研究所

出　　处：《中华微生物学和免疫学杂志》2010年第2期104-109,共6页Chinese Journal of Microbiology and Immunology

基　　金：卫生部基金项目资助（wkj2006-02-206）

摘　　要：目的稳定表达鼠IFN—λ2并对其生物学活性进行研究。方法用水疱口炎病毒（vesicular stomatitis virus，VSV）刺激小鼠脾脏细胞，克隆mIFN—λ2全长基因，构建真核表达载体PCAGG-EGFP-mIFN-λ2，并在CHO细胞稳定表达，且在小鼠黑色素瘤B16细胞上进行抗病毒活性测定；构建MDBK-Mxp—Luc细胞系诱导Mx1抗病毒蛋白产生。结果pMD18-T-mIFN—λ2双酶切鉴定，出现582bp大小的条带，成功构建了PCAGG—EGFP—mIFN—λ2真核表达载体；稳定表达mIFN—λ2 CHO的细胞株分泌的上清中mIFN-λ2蛋白在B16细胞上的抗病毒活性为10^4AU／ml；mIFN-λ2蛋白诱导鼠Mx1抗病毒蛋白的表达，9～12h达高峰，24h后消失（P〈0．05）。结论建立了稳定表达mIFN—λ2的CHO细胞株，其分泌型mIFN—λ2蛋白具有明显的抗病毒活性。Objective To express mouse IFN-λ2 stably and study its biological activity. Methods Full-length of mIFN-λ2 eDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by ve- sicular stomatitis virus（VSV） ancl then subcloned to eukaryotie expressing vector PCAGG-EGFP. The recombinant 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- k2 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 10^4AU/ml. The mIFN-λ2 protein 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 1 2 hours achieved the peak, 2 4 hours vanished. Conclusion Gene cloning of mIFN-λ2 was successful. The eukaryotic expressing vector of mIFN-λ2 was constructed successfully 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.

关 键 词：mIFN-λ2 基因克隆 稳定表达 抗病毒活性 Mx1蛋白 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]