人λ干扰素在BHK21中表达及生物学活性探究.docVIP

人λ干扰素在BHK21中表达及生物学活性探究 　 作者：严玉兰, 刘洋, 曹文雁, 步雪峰 步志高, 郑金旭 【摘要】 目的: 研究HuIFNλ1和HuIFNλ2真核细胞重组表达及其生物学活性。方法: 用水疱性口炎病毒（VSV）刺激人宫颈癌HeLa细胞, 用RTPCR克隆HuIFNλ1和HuIFNλ2基因, 与PCAGGEGFP真核表达载体连接, 构建重组体PCAGGHuIFNλ1和PCAGGHuIFNλ2并进行鉴定, 后在BHK21细胞进行表达, 采用VSV*GFP于人肺腺癌A549上进行表达产物抗病毒活性测定; 并通过已构建的MDBKMxpLuc细胞系对HuIFNλ1和HuIFNλ2诱导MxA抗病毒蛋白产生的特性进行研究。结果: HuIFNλ1PMD18T Vector和HuIFNλ2PMD18T Vector的SacⅠ、 XhoⅠ和SacⅠ和SalⅠ双酶切鉴定, 均出现610 bp大小的带, 测序示与GenBank上报道的序列完全一致。PCAGGHuIFNλ1活性104 IU/mL, PCAGGHuIFNλ2活性为102 IU/mL, 且PCAGGHuIFNλ1诱导Mx抗病毒蛋白的表达一定时间内随时间延长表达不断增强, 9～12 h达高峰, 24 h后消失（Plt;0.05）。结论: 成功构建了PCAGGHuIFNλ1PCAGGHuIFNλ2 的真核表达载体并在BHK21细胞中得到表达, 其抗病毒活性与诱导Mx抗病毒蛋白密切相关。 【关键词】 干扰素λ; 基因转染; 生物活性 ［Abstract］ AIM: To construct the eukaryotic expressing vector PCAGG HuIFNλ1 and PCAGGHuIFNλ2 and to study the biological activity of HuIFNλ1and HuIFNλ2. METHODS: The cDNA fragment encodding HuIFNλ1 and HuIFNλ2 was amplified from the total RNA extracted from virusinduced HeLa cells by RTPCR. Then it was cloned into the eukaryotic expressing vector PCAGGEGFP. The recombinant was transfected into BHK21 cells. VSV*GFPA549 system was used to measure the antivirus activity.The constructed cell line MDBKMxpLuc was used to study the characteristics of MxA protein induced by the products of PCAGGHuIFNλ1 and PCAGGHuIFNλ2. RESULTS: The recombinant vector HuIFNλ1PMD18T Vector was enzymed by SacⅠand XhoⅠ while HuIFNλ2PMD18T Vector was enzymed by SacⅠ and SalⅠ. The fragments were both 610 bp and they were consistent with nucleotide sequences reported in GenBank. The antivirus activity of protein expressed by PCAGGHuIFNλ1 and PCAGGHuIFNλ2 was 104 IU/mL and 102 IU/mL, respectively. The protein expressed by PCAGGHuIFNλ1 and PCAGGHuIFNλ2 induced the expression of the antivirus protein MxA. The expression of protein MxA induced by PCAGGHuIFNλ1 increased with the passage of time, reaching the peak during 9 to 12 hours and disappearing in 24 hours. CONCLUSION: The e