摘要
人巨细胞病毒(human cytomegalovirus,HCMV)在自然界中普遍存在,g B是该病毒表面的一种重要糖蛋白,其AD1序列是主要的中和抗体表位之一。首先以HCMV基因组为模板,通过PCR技术扩增出g B基因的一个片段,该片段删除了跨膜区和胞内区,仅保留AD1中和抗体表位,然后被连接到哺乳动物细胞表达质粒p SGHV0上,与dhfr基因通过电击转染至CHO/dhfr-细胞,继而用60 nmol/L MTX筛选稳定表达细胞株,用ELISA和Western blot检测AD1重组蛋白的表达,最后用Ni2+-NTA亲和层析进行纯化。获得了真核细胞表达的重组融合蛋白,表达量约为10 mg/L,蛋白纯度可达到80%以上,为HCMV表面糖蛋白g B功能的进一步研究以及基因工程疫苗的开发奠定了基础。
Human cytomegalovirus （HCMV） is commonly found in nature. HCMV gB is an important surface glycoprotein, and its AD1 sequence is one of the major neutralizing epitopes. First, the truncated gB gene containing the AD1 sequence but without the transmembrane and intracellular domains was amplified by PCR using HCMV genomic as template. Then the HCMV gB/AD1 gene was cloned into mammalian cell ex- pression vector pSGHV0, and electroporated with dhfr gene into CHO/dhfr- cells. Stable cell lines were fur- ther screened with 60 nmol/L MTX, and the expression of recombinant AD1 protein was detected by ELISA and Western blot. Finally the fusion protein was purified by Ni2＋-NTA affinity chromatography. The recombi- nant fusion protein was successfully obtained with expression level of about 10 mg/L and purity of over 80%, which can laid solid foundation for further studies of HCMV gB function and genic engineering vaccine research in the future.
出处
《生命科学研究》
CAS
CSCD
2015年第5期402-409,共8页
Life Science Research
