神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用

畜牧兽医学报

神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用

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*

（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）

收稿日期:2012-07-08 出版日期:2013-01-23 发布日期:2013-01-23
通讯作者: 李碧春， E-mail: yubcli@yzu.edu.cn
作者简介:傅德智（1987-），男，山西灵石人，硕士生，主要从事动物胚胎工程与遗传工程的研究，E-mail: wlhuanxinang@gmail.com
基金资助:
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

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

FU De-zhi¹, ZHANG Ya-ni¹, CHEN Hao², LI Wei¹, ZHENG Meng-meng¹, WANG Dan¹, ZHANG Zhen-tao¹, SHI Qing-qing¹, LI Bi-chun^1*

(1. Institute of Animal Breeding and Genetics, Provincial Key Laboratory of Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; 2. The First affiliated hospital of Soochow University, Suzhou 215006, China)

Received:2012-07-08 Online:2013-01-23 Published:2013-01-23

摘要/Abstract

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72 h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48 h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。

Abstract:

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 transfected 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 Mx gene were derived from pcDNA3.0-NA, pcDNA3.0-Mx plasmid by PCR, respectively. NA cDNA fragment and Mx 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 transfected into CEF cells，RT-PCR and the micro-cell inhibition test were used to test the antiviral activity for NDV (Newcastle disease virus). The restriction endonuclease digestion and sequencing results suggested that co-expression vector pVITRO₂-Mx-NA was constructed successfully, the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. Recombinant proteins of Mx and NA protected CEF cells from NDV infection during 0-72 hours of incubation, 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 significantly delayed the NDV infection time of CEF cells, the difference was statistically significant (P<0.05). 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.

中图分类号:

Q784

傅德智，张亚妮，陈昊，李伟，郑蒙蒙，王丹，张振韬，施青青，李碧春. 神经氨酸酶基因联合抗黏液病毒基因的抗病毒作用[J]. 畜牧兽医学报, doi: .

FU De-zhi, ZHANG Ya-ni, CHEN Hao, LI Wei, ZHENG Meng-meng, WANG Dan, ZHANG Zhen-tao, SHI Qing-qing, LI Bi-chun. Antiviral Activities of Neuraminidase Gene Combining with Chicken Mx against Newcastle Disease Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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