【摘要】 从HepG2细胞中提取总RNA,经RT-PCR扩增α-2,6唾液酸转移酶基因,构建于pMD-18T克隆载体中,经序列测定后与GenBank中报道的已知序列比对完全一致,再将已知目的序列插入原核表达载体pET-28a(+)中。将构建正确的原核表达载体转化表达受体菌BL21(DE3)中,IPTG诱导其进行表达并鉴定目的蛋白,对鉴定正确的目的蛋白复性后经N i2+亲和层析柱纯化获得高纯度的α-2,6唾液酸转移酶蛋白。然后用霍乱弧菌神经氨酸酶处理健康鸡红细胞,清除鸡红细胞表面所有类型的唾液酸寡糖链;再用表达的α-2,6唾液酸转移酶以CMP唾-液酸作为底物在霍乱弧菌神经氨酸酶处理的鸡红细胞表面标记上SAα2,6 Gal受体。将标记有SAα2,6Gal受体的鸡红细胞应用微量血凝试验和流式细胞仪进行检测,以验证所表达蛋白的生物活性。结果表明,原核表达的α-2,6唾液酸转移酶具有较好的生物活性。更多还原

【Abstract】 To analyse the bioactivity of α-2,6 sialyltransferase protein,the total RNA was extracted from HepG2 cell and the α-2,6 sialyltransferase gene included an open reading frame coding for 406 amino acids was amplified using RT-PCR.Then the amplified products were inserted into pMD-18T vector.After sequenced,the target DNA fragment was cloned into pET-28a(+) vector.The recombinant plasmid was transformed into E.coli BL21(DE3),and the target protein was expressed after induction with 0.5 mmol/L isopropy-β-D-thiogalactoside(IPTG) at 37℃ for 4 hours.The expressed protein was purified by Ni2+-NTA affinity chromatography.Then all of the sialic acid was removed from chicken red blood cells(CRBCs) using vibrio cholerae neuraminidase(VCNA).Subsequently,resialylation was performed using α-2,6 sialyltransferase and 1.5 mmol/L CMP-sialic acid.To identify the receptor specificity of CRBCs using hemagglutination assay of influenza virus,and flow cytometry(FCM).The results showed that purified α-2,6 sialyltransferase protein has biological characteristics,which might be a candidate for clinical diagnosis for H5N1 avian influenza virus.更多还原