CAJ | 학술논문

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582 bp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10~4 AU/ml;mIFN-λ2蛋白诱导鼠Mx1抗病毒蛋白的表达,9～12 h达高峰,24 h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,且与诱导Mx1抗病毒蛋白密切相关.
목적 은정표체서IFN-λ2병대기생물학활성진행연구.방법 용수포구염병독(vesicular stomatitis virus,VSV)자격소서비장세포,극륭mIFN-λ2전장기인,구건진핵표체재체PCAGG-EGFP-mIFN-λ2,병재CHO세포은정표체,차재소서흑색소류B16세포상진행항병독활성측정;구건MDBK-Mxp-Luc세포계유도Mx1항병독단백산생.결과 pMD18-T-mIFN-λ2쌍매절감정,출현582 bp대소적조대,성공구건료PCAGG-EGFP-mIFN-λ2진핵표체재체;은정표체mIFN-λ2 CHO적세포주분비적상청중mIFN-λ2단백재B16세포상적항병독활성위10~4 AU/ml;mIFN-λ2단백유도서Mx1항병독단백적표체,9～12 h체고봉,24 h후소실(P<0.05).결론 건립료은정표체mIFN-λ2적CHO세포주,기분비형mIFN-λ2단백구유명현적항병독활성,차여유도Mx1항병독단백밀절상관.
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.