猪传染性胃肠炎病毒S基因A抗原位点的克隆及原核表达载体的构建

摘要/Abstract

摘要： 【研究目的】对猪传染性胃肠炎病毒S基因A抗原位点进行克隆和原核表达载体的构建。【方法】参考GenBank上公布的TGEV S基因A抗原位点序列，应用Primer6.0设计一对含酶切位点的引物，用于RT-PCR扩增A抗原位点目的片段，将扩增产物连接于paesy-T克隆载体上构建克隆载体，用EcoR I和Xhol I对表达载体PET-32a（+）和重组质粒进行酶切，将酶切产物亚克隆至PET-32a（+）多克隆位点上，连接、转化至BL21(DE3)，构建A位点的原核表达载体，并对阳性重组质粒进行酶切、PCR和测序鉴定。【结果】所扩增的目的片段的大小为534bp，与原核表达载体连接后，经核苷酸及推导的氨基酸序列分析表明，该基因与其它猪传染性胃肠炎病毒相应基因具有很高的同源性，说明成功地构建了TGEV S 基因A抗原位点的原核表达载体。【结论】TGEV S 基因A抗原位点原核表达载体的成功构建，填补了中国国内单独针对此位点进行研究的空白，也为TGEV诊断方法的建立提供良好的技术基础。

关键词: 樟子松实生苗针叶, 樟子松实生苗针叶, 温度, 光周期, 抗寒性, 加性影响

Abstract: 【OBJECTIVE】To clone the site A of S gene of transmissible gastroenteritis virus and to construct the prokaryotic expression vectors of site A.【METHOD】The target gene of the site A of S gene was amplified respectively by RT-PCR with a pair of primers, which was designed according to the published sequence of TGEV S gene with primer 6.0 software. The amplified gene was cloned into the vector peasy-T , Then the gene and the vector PET-32a（+）was digested by EcoR I and Xhol I. The recycled genes were inserted into the multiple cloning sites of the vector PET-32a（+），The recombinant plasmid was transformed into BL21(DE3) strain and the positive plasmid was identified by restriction enzyme, PCR and sequencing.【RESULTS】The results showed that the length of target gene was 534bp and the nucleotide had a relatively high homology with the corresponding gene from other TGEV strains according to the nucleotide and deduced amino acid sequences. The site A gene were cloned into the vector peasy-T and the recombinant expression vectors were constructed successfully.【CONCLUSION】The construction of recombinant expression vectors filled up a gap of the study on site A of S gene in China. It also could provide a technical basis for the diagnosis of TGEV.

张春叶,沈红,张莉,李焕荣,路苹. 猪传染性胃肠炎病毒S基因A抗原位点的克隆及原核表达载体的构建[J]. 中国农学通报, 2008, 24(7): 11-16.

Zhang Chunye, Shen Hong, Zhang Li, Li Huanrong, Lu Ping. Cloning and the Construction of Prokaryotic Expression of
the Site A of S Gene of Transmissible Gastroenteritis Virus
[J]. Chinese Agricultural Science Bulletin, 2008, 24(7): 11-16.

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