人λ-干扰素在BHK-21中的表达及生物学活性的研究

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(P＜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.

Human lamada-interferon expressed in BHK-21 cell line and its bioactivity

AIM: To construct the eukaryotic expressing vector PCAGG -HuIFN-lambda1 and PCAGG-HuIFN-lambda2 and to study the biological activity of HuIFN-lambda1and HuIFN-lambda2. METHODS: The cDNA fragment encodding HuIFN-lambda1 and HuIFN-lambda2 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-lambda1 and PCAGG-HuIFN-lambda2. RESULTS: The recombinant vector HuIFN-lambda1-PMD18-T Vector was enzymed by Sac I and Xho I while HuIFN-lambda2-PMD18-T Vector was enzymed by Sac I and Sal I. 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-lambda1 and PCAGG-HuIFN-lambda2 was 10(4) IU/mL and 10(2) IU/mL, respectively. The protein expressed by PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2 induced the expression of the anti-virus protein MxA. The expression of protein MxA induced by PCAGG-HuIFN-lambda1 increased with the passage of time, reaching the peak during 9 to 12 hours and disappearing in 24 hours. CONCLUSION: The eukaryotic expressing vector of PCAGG-HuIFN-lambda1 and PCAGG-HuIFN-lambda2 has been successfully constructed and transiently expressed in BHK-21 cells. The anti-virus activity of the products is closely correlated with inducing the expression of anti-virus protein MxA.