摘要
为了构建猪α-干扰素(PoIFN-α)的真核表达系统,鉴定其生物学活性,分离猪的外周血淋巴细胞,提取总RNA,利用RT-PCR技术扩增猪α-干扰素基因序列,克隆至pcDNA3.1(+)载体,并转染BHK-21细胞。通过G418筛选获得稳定转染的细胞株,采用real-time PCR及ELISA测定PoIFN-α基因在BHK-21细胞内的表达,利用淋巴细胞增值试验(MTT)检测表达蛋白的生物学活性。结果表明:稳定整合PoIFN-α基因的BHK-21细胞系其PoIFN-αmRNA的转录效率比阴性对照组提高1.52倍,蛋白表达量显著提高1.35倍。MTT试验表明真核表达PoIFN-α淋巴细胞增值活性与阴性对照组相比差异显著(P<0.01),表明BHK-21细胞表达的PoIFN-α具有良好的生物学活性,为开发新型抗病毒制剂奠定基础。
To construct expression system of porcine interferon-α and identify its biological activity. The porcine interferon-α gene （PoIFN-α） was amplified from the total RNA of porcine peripheral blood lymphocytes by RT-PCR and inserted into pcDNA3. 1（＋） vector,which was ultimately transfected into BHK-21 cells. A BHK-21 cell line expressing PolFN-α was established by G418 screening and RT-PCR identification. Gene expression level and activity of PoIFN-α in BHK-21 cells were examined by Real-time PCR, ELISA, and lymphocyte proliferation test （MTT）, respectively. The results showed that mRNA expression level and activity of PoIFN-α expressed in BHK-21 cells were 1.52 and 1.35 times higher than negative control cells,respectively. Furthermore,MTT revealed PoIFN-α produced by eukaryotic cells was able to significantly stimulate proliferation of lympho- cyte （p〈0.01） ,indicating that the recombinant PoIFN-α exhibited a good biological activity. The present study provides a foun-dation for further development of new antiviral agents.
出处
《石河子大学学报（自然科学版）》
CAS
2013年第2期182-186,共5页
Journal of Shihezi University(Natural Science)
基金
国家自然科学基金项目(31260534)
国家青年科学基金项目(31201800)
