人λ-干扰素在BHK-21中的表达及生物学活性的研究

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

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-TVector和HuIFN-λ2-PMD18-TVector的SacⅠ、XhoⅠ和SacⅠ和SalⅠ双酶切鉴定,均出现610bp大小的带,测序示与GenBank上报道的序列完全一致。PCAGG-HuIFN-λ1活性104IU/mL,PCAGG-HuIFN-λ2活性为102IU/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,9～12h达高峰,24h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。

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.VSVGFP-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 eukaryotic expressing vector of PCAGG-HuIFN-λ1 and PCAGG-HuIFN-λ2 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.