抗口蹄疫病毒相关基因的筛选及分析

口蹄疫是一种烈性传染病，其广泛流行给社会造成了巨大经济损失。为了研究口蹄疫灭活疫苗免疫的分子机制，同时也为抗口蹄疫病毒药物的研制奠定基础，本研究应用mRNA差异显示技术，以PK-15细胞为材料，系统比较了口蹄疫疫苗刺激组（A组）和正常的PK-15细胞（B组）的基因表达情况，回收差异片段，经二次扩增并纯化后，得到30条ESTs。将30条ESTs采用以地高辛标记的反向Northern点杂交鉴定，将阳性条带克隆测序，筛选出8条ESTs，编号E1~E8，应用BLASTn工具将8条ESTs对核酸数据库nr和dbEST中所有序列进行了同源性分析，其中E1，E2分别与猪的热休克蛋白基因、猪的MHCⅠ类基因同源，序列相似性都达到100%。E3，E4，E5，E7分别与已有核酸数据库中的基因克隆或EST具有较高同源性，为已知的EST，但功能未知；E6，E8在数据库中没有发现与其相似性较高的序列，为新的EST。应用数据库资源将E5、E7进行电子延伸后，将延伸序列进行开放阅读框分析，又经TBLASTx分析发现E7蛋白质序列与猪的精氨酸酶Ⅰ类蛋白序列有很高同源性。将E1、E2、E4、E5序列进行了基因表达谱分析，对E6、E8用BLASTx工具对非冗余蛋白质数据库nr进行了相似性搜索，在其他物种中找到了相似的基因序列。本研究筛选出的热休克蛋白基因、MHCⅠ类基因、精氨酸酶Ⅰ类基因和其他未知功能基因可以作为抗口蹄疫病毒研究中的侯选基因，其具体的功能有待今后进一步研究。

Screening and Analysis of Anti-FMD Related Genes

Abstract:Enormous economic loses were caused by FMD which was a kind of virulent infections disease.This study was carried out to research the molecular immune mechanism of inactivated FMD vaccines, lay the foundations for invention of anti-FMDV medicine. In the research, mRNA differential display method was used to compare gene expression between PK-15 cells stimulated by Swine Foot and Mouth Disease and normal controls. 30 ESTs were obtained by extraction , amplification and purification. The 30 ESTs were identified by reverse northern dot bloting .8 positive DNA fragments were cloned and sequenced.All 8 ESTs were compared with nucleotide sequences deposited in the nr database and dbEST database of GenBank via BLASTn tool. E1 was found highly similar to Heat shock 60kDa　protein 1, E2 was found highly similar to Sus scrofa MHC class I SLA genomic region, haplotype H01,clone. E7 were extended and separated by searching database. The ORF database were used to analyse the extened bands. The result indicated that E7 was highly similar to arginase I of Sus scrofa. heat shock 60kDa protein 1, Sus scrofa MHC class I SLA genomic region haplotype H01 clone, arginase, type I of Sus scrofa and the other genes with unknown functions were regarded as candidate genes in anti-FMDV research, and their specific functions need to be explored in further study.