摘要
为鉴定BHK-21细胞中稳定表达猪白细胞介素-2(PoIL-2)的活性,本研究利用RT-PCR技术从猪的淋巴细胞中克隆PoIL-2基因,将其连接于pcDNA3.1载体中,并转染BHK-21细胞。经过G418筛选及RT-PCR鉴定,获得稳定表达PoIL-2基因的BHK-21细胞系。采用实时定量PCR及ELISA测定PoIL-2在BHK-21细胞内的表达,同时以淋巴细胞增殖试验(MTT)检测PoIL-2蛋白活性。试验结果表明稳定整合PoIL-2基因的BHK-21细胞系其PoIL-2mRNA的转录效率是阴性对照组1.43倍,蛋白表达量是阴性对照组1.29倍。MTT试验表明真核表达的PoIL-2促淋巴细胞增殖活性与阴性对照组相比差异极显著(p<0.01),表明BHK-21细胞表达的PoIL-2具有良好的生物学活性。本研究为开发新型基因工程疫苗佐剂和抗病毒制剂奠定了基础。
To express porcine interleukin-2 （PoIL-2） in eukaryotic cells and evaluate its biological activity, the PoIL-2 gene was amplified from pig lymphocyte by RT-PCR and inserted into pcDNA3.1 vector, which was ultimately transfected into BHK-21 cells. A BHK-21 cell line expressing PoIL-2 gene was established by G418 screen and RT-PCR identification. Gene expression level and activity of PoIL-2 in BHK-21 cells was examined by Real-time PCR, ELISA, and lymphocyte proliferation test （MTT）, respectively. The result showed that mRNA expression level and activity of PoIL-2 expressed in BHK-21 cells was 1.43 and 1.29 times higher than negative control cells, respectively. Furthermore, MTT revealed PoIL-2 produced by eukaryotic cells was able to significantly stimulate proliferation of lymphocyte （p0.01）, indicating that the recombinant PoIL-2 exhibits a good biological activity. The present study provided foundation for further investigation on developing new genetic engineering adjuvant vaccine and antiviral agents.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第5期378-382,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
转基因生物新品种培育国家重大专项(2009ZX08005-003B)
