摘要
为了研究密码子偏性对痘苗病毒载体表达效率的影响,分别采用痘苗病毒及其宿主细胞的优势密码子对绿色荧光蛋白基因进行改造,利用荧光、Western blot和FCM等方法分析其在痘苗病毒载体系统的表达水平。结果显示,全部采用痘苗病毒优势密码子(富含A+T)和全部采用宿主细胞优势密码子(富含G+C),以及部分使用宿主细胞优势密码子的三种绿色荧光蛋白基因都能够有效表达,表达水平相近,表明痘苗病毒载体对目的基因密码子的使用具有很好宽容性。为了探讨这种宽容性的机理,分别利用在胞核内和在胞浆内转录的质粒载体对不同密码子偏性的绿色荧光蛋白基因进行表达分析。结果显示,胞核内转录目的基因的pcDNA3质粒载体能有效表达富含G+C的绿色荧光蛋白基因,不能有效表达富含A+T的绿色荧光蛋白基因,而胞浆内转录目的基因的pSCA质粒载体能同样有效表达上述不同密码子偏性的目的基因。这些结果表明,位于胞浆内的富含A+U的转录产物能够有效表达,细胞核内生成的富含A+U的转录产物可能受核膜屏障或其它核内因素影响而不能有效表达。因此,胞浆内繁殖的特性是痘苗病毒载体具有密码子宽容性的主要原因。此研究为痘苗病毒载体和常用真核表达载体的选择使用提供了重要实验依据。
To determine the relationship between codon bias and the gene expression efficiency in vaccinia virus vector, green fluorescent protein gene was recoded and synthesized according to the preferred codons by human and vaccinia virus, respectively. Then each of those synthesized genes was used to construct recombinant vaccinia virus （rVV）. Immunofluorescence, Western blot and flow cytometry analyses were used to analyze the expression levels of synthetic genes in rVV-infected HeLa cells. It was also analyzed on the expression levels of the three green fluorescent protein genes with different codon bias in traditional plasmid vector pcDNA3 （transcribing target gene in nucleus） and SFV-based plasmid vector pSCA （transcribing target gene in cytoplasm）. The results showed that all of these three target genes had expressed efficiently in vaccinia virus vector and the codon bias in those target genes just had slight impact on the expression efficiency. However, the G ＋ C rich green fluorescent protein genes in pcDNA3 plasmid vector, including mGFP gene （G ＋ C content is 51.5 % ） and EGFP gene （G＋ C content is 61.8% ） were expressed efficiently, but the A ＋ T rich green fluorescent protein gene VGFP （ A ＋ T content is 73.1% ） exhibited very poor expression. In constract, all green fluorescent protein genes in pSCA plasmid vector had efficient and comparable expression in suitable host cells. These results demonstrated that vaccinia virus vector has a broad compatibility to the codon usage of target genes. The mechanism of this broad compatibility is mainly related to the cytoplasmic transcription of vaccinia virus. Our study provides very useful information on choosing vaccinia virus vector and other common eukaryotic vectors.
出处
《病毒学报》
CAS
CSCD
北大核心
2006年第5期350-357,共8页
Chinese Journal of Virology
基金
CIPRA项目(1U19A15191501)
863项目(2003AA219080)
关键词
密码子偏性
痘苗病毒载体
基因表达
codon bias
vaccinia virus vector
expression efficiency
