神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用  

Antiviral Activities of Neuraminidase Gene Combining with Chicken Mx against Newcastle Disease Virus

作　　者：傅德智[1] 张亚妮[1] 陈昊[2] 李伟[1] 郑蒙蒙[1] 王丹[1] 张振韬[1] 施青青[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009 [2]苏州大学第一附属医院,苏州215006

出　　处：《畜牧兽医学报》2013年第1期78-86,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省高校自然科学重大基础研究项目资助(08KJA230001);高等学校博士学科点专项;"六大人才高峰"资助项目

摘　　要：拟构建神经氨酸酶(NA)基因和抗黏液病毒(Mx)基因双基因真核共表达载体,并检测基因表达以及转染鸡成纤维(CEF)细胞后的抗病毒效果。克隆了NA基因和突变型Mx基因的cDNA;分别定向插入真核细胞双顺反子载体pVITRO2中,酶切和测序鉴定双基因共表达载体pVITRO2-Mx-NA的正确性。用pVITRO2-Mx-NA转染NIH-3T3细胞,通过RT-PCR和间接免疫荧光(IFA)方法检测目的基因的表达。随后用pVITRO2-Mx-NA转染CEF细胞,利用RT-PCR及微量细胞病变抑制法测定重组蛋白抗新城疫病毒(NDV)的效果。结果:酶切和测序分析表明成功构建了双基因真核共表达载体pVITRO2-Mx-NA,在转染后的NIH-3T3和CEF细胞中同时检测到了NA和Mx基因的表达。联合基因表达的重组蛋白可保护CEF细胞在孵育的72h内免受NDV的感染,而转染了突变型Mx或者NA单基因真核表达载体的CEF细胞在孵育的48h内亦未受到NDV的侵染;与单基因转染组相比,联合基因转染组明显延迟NDV感染CEF细胞的时间,差异显著(P<0.05)。构建的双基因真核表达载体pVITRO2-Mx-NA转染CEF细胞后,其表达的重组蛋白Mx-NA在细胞水平上具有协同延缓NDV感染的能力,且优于单个基因。This experiment was conducted to build the neuraminidase （NA） gene and anti-mucus virus （Mx） dual eukaryotic gene co-expression plasmid,and detect the gene expression in trans- fected mouse fibroblasts （NIH-3T3） cells, and to investigate the influence of the recombinant plasmid on the chicken fibroblasts （CEF） cells. The cDNA fragment of NA and mutant Mac gene were derived from pcDNA3. 0-NA, pcDNA3. 0-Mx plasmid by PCR, respectively. NA cDNA fragment and Mac cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to construct the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenced, then the mouse fibroblasts （NIH-3T3） cells were transfected. The expression of genes in pVITRO2-Mx-NA were identified by RT-PCR and indirect immunofluorescence assay （IFA）. Then the recombinant plasmid was transfeeted into CEF cells,RT-PCR and the micro-cell inhibition test were used to test the antivi- ral activity for NDV （Newcastle disease virus）. The restriction endonuclease digestion and se- quencing results suggested that co-expression vector pVITRO2-Mx-NA was constructed success- fully, the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. Recombi- nant proteins of Mx and NA protected CEF cells from NDV infection during 0-72 hours of incuba- tion, the mutagenesis Mx or NA protein protected CEF cells from NDV infection during 0-48 hours of incubation Compared with single-gene transfection group,co-transfection group signifi- cantly delayed the NDV infection time of CEF cells, the difference was statistically significant （P d0.05）. The recombinant eukaryotic expression vector pVITRO2-Mx-NA was constructed and expressed in CEF cell successfully, which would contribute to delaying the infection of NDV in cell level, it revealed the co-transfection of the combined genes is more powerful than single one, they had synergistic effects.

关 键 词：NA MX 双基因表达载体 抗病毒活性 抗NDV 

分 类 号：Q784[生物学—分子生物学]