摘要
目的表达纯化一种新型重组人干扰素Epsilon(rhIFNε155ser),并对它的生物学特性进行初步研究。方法人工合成rhIFNε155ser的全基因序列,并按照大肠埃希菌密码子嗜性作适当改造,然后构建到原核表达载体pBV220,在大肠埃希菌DH5α中表达。将表达的包涵体蛋白纯化复性后,对最终产物的抗病毒活性、细胞生长抑制活性和NK细胞刺激活性进行鉴定。同时,利用芯片技术,从基因的水平对rhIFNε155ser的生物机制进行初步的了解。结果rhIFNε155ser以包涵体的形式表达,经纯化蛋白纯度可达95%。复性后rhIFNε155ser在WISH抗VSV系统中的抗病毒活性可达6×105IU/mg。在100～1000pg/ml下rhIFNε155ser具有剂量依赖性的抗增殖活性,而NK细胞刺激活性无剂量依赖性。基因芯片扫描发现,22278个位点中,有283个基因显著上调,另外1894个显著下降。结论成功地表达了rhIFNε155ser,发现此IFN具有一定的抗病毒、抗增殖和NK细胞刺激活性。芯片技术可进一步拓展对IFN的功能研究。
Objective To construct a novel recombinant rhIFN-ε155ser and study its biological activities, Methods The v, hole sequence of rhIFN-ε was artificially synthesized and some codons were altered according to the preferred codon using of E. coli. The sequence was cloned into plasmid vector pBV220 to express in E. coli DH5α. After purification and re-folding of rhIFN-ε155ser inclusion body, the final product was tested for its biological activities, including anti-viral, anti-proliferative and NK cell enhancing activities. At the same time, by using DNA microarray bioehips, the gene expression patterns in the rhIFN-ε155ser and rhIFN-α2b treated cells were compared and analyzed. Results The re-built rhIFN-ε155ser sequence was expressed in E. coil as a form of inclusion body. After purified and re-folded, the rhIFN-ε155ser protein reached a purity of above 95%. The rhIFN-ε155ser protein had a specific anti-viral activity of about 6 × 10^5IU/ mg in WISH / VSV system. Its anti-proliferative activity and NK cell enhancing activities in vitro seemed to be lower than that of rhIFN-α2b. Data obtained from microarray bioehips indicated that there were 283 pieces increasing 2 folds and 1489 pieces decreasing 2 folds among totally 22 278 pieces of human genes were found in the rhIFN-ε155ser treated cells ; more changes in gene expression pattern were detected in the rhIFN-α treated cells. Conclusion A novel recombinant rhIFN-ε155ser was constructed, which belonged to type I interferon. The biological activities of rhIFN-ε155ser were compared with rhIFN-α2b. The changes of gene expression pattern in the interferon treated cells were detected, analyzed and discussed.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2005年第3期227-231,共5页
Chinese Journal of Experimental and Clinical Virology
