绵羊Nectin 4基因的克隆与表达

发布时间：2018-05-11 03:00

本文选题：Nectin + 巢氏PCR　；参考：《中国兽医科学》2016年01期

【摘要】：采用巢式PCR方法获得未见报道的绵羊Nectin4基因。根据GenBank上登录的牛的Nectin4mRNA序列信息设计并合成10对引物;从绵羊肺支气管组织中提取总RNA,采用随机引物和Oligo(dT)将其反转录为cDNA;利用巢式PCR原理和PCR引物错配导致非特异反应的特点,改变常规PCR反应条件,将10对引物分为2组,通过两轮PCR即获得大小片段相符的片段;TA克隆后测序证实已成功获得绵羊Nectin4基因的完整ORF,证明本方法可作为一种获取未知基因的方法,较RACE等其他传统技术有明显优势;构建了重组表达质粒pCMV-Myc-Nectin 4,在CHO细胞的细胞质中获得高效表达,经Western-blot分析,表达的Nectin 4蛋白的大小与预期符合。绵羊Nectin4基因的成功扩增为后续对其在病毒入侵中的功能进行研究奠定了基础。
[Abstract]:Sheep Nectin4 gene was obtained by nested PCR method. Ten pairs of primers were designed and synthesized according to the information of Nectin4mRNA sequence of cattle registered on GenBank; total RNAs were extracted from sheep lung bronchi tissue and reversely transcribed into cDNAusing random primers and OligodT.The nested PCR principle and the characteristics of PCR primer mismatch led to nonspecific reactions. Ten pairs of primers were divided into 2 groups by changing the conventional PCR reaction conditions. The complete ORF of sheep Nectin4 gene has been obtained successfully by two rounds of PCR sequencing. It is proved that this method can be used as a method to obtain unknown genes, which has obvious advantages over other traditional techniques such as RACE. The recombinant expression plasmid pCMV-Myc-Nectin _ 4 was constructed and highly expressed in the cytoplasm of CHO cells. The size of the expressed Nectin _ 4 protein was in accordance with the expectation by Western-blot analysis. The successful amplification of sheep Nectin4 gene laid a foundation for further study on its function in virus invasion.
【作者单位】：河南科技学院动物科学学院;中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室;甘肃农业大学动物科学技术学院;中国农业科学院北京畜牧兽医研究所动物营养国家重点实验室;
【基金】：现代农业产业技术体系建设专项(CARS-40-10) 公益性行业(农业)科研专项经费项目(201103008) 河南省高等学校重点科研项目(15A230002)
【分类号】：S826;S852.65

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