赤羽病病毒G1蛋白生物信息学分析及截短基因的真核表达  

Bioinformatics analysis of G1 protein of Akabane virus and eukaryotic expression of its truncated genes

作　　者：胡世享 叶玲玲 肖妍 卓娜 陈朝林 汪琳 蒲静 陈培富[1] 艾军 HU Shixiang;YE Lingling;XIAO Yan;ZHUO Na;CHEN Chaolin;WANG Lin;PU Jing;CHEN Peifu;AI Jun(Yunnan Agricultural University,Kunming 651000,China;Kunming Customs Technology Center,Kunming 651000,China;Tianjin Customs Animal,Plant and Food Testing Center,Tianjin 300041,China;China Customs Science and Technology Research Center,Beijing 100026,China)

机构地区：[1]云南农业大学,云南昆明651000 [2]昆明海关技术中心,云南昆明651000 [3]天津海关动植物与食品检测中心,天津300041 [4]中国海关科学技术研究中心,北京100026

出　　处：《畜牧与兽医》2023年第10期61-69,共9页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFC2601605)。

摘　　要：为得到赤羽病病毒(AKV)G1蛋白的截短表达产物作为诊断抗原,通过PCR扩增、酶切、连接、转化等技术手段,成功克隆出优势抗原结合表位区Thr189-Val 397对应的目的基因G1-2。将目的基因克隆至昆虫杆状病毒表达载体pFastBac HTB,将该重组质粒转化含有杆状病毒穿梭载体的DH10Bac感受态细胞,得到重组穿梭质粒Bacmid-AKV-G1-2,用Cellfectin ReagentⅡ介导转染sf9细胞,获得重组蛋白,Western blot法检测和分析重组蛋白表达情况。软件分析结果表明189~397 aa肽段为亲水性蛋白,存在跨膜区,该蛋白存在多个潜在的磷酸化位点,具有丰富的潜在抗原表位,属于优势抗原肽段,选取该肽段进行截短真核表达,以AKV抗体阳性血清进行Western blot鉴定,重组蛋白能被特异性识别,出现预期蛋白反应条带,表明G1-2蛋白成功表达,分子量约为27 kDa。本研究为进一步开展截短表达产物为抗原的赤羽病病毒诊断试剂研制奠定了基础。In order to obtain the truncated expression product of Akabane disease virus(AKV)G1 protein as a diagnostic antigen,the tar-get gene G1-2 corresponding to the dominant antigen-binding epitope Thr189-Val397 was successfully cloned by PCR amplification,diges-tion,ligation,transformation and other technical means.The target gene was cloned onto the insect baculovirus expression vector pFastBac HTB,the recombinant plasmid was transformed into DH10Bac-competent cells containing a baculovirus shuttle vector to obtain the recombi-nant shuttle plasmid Bacmid-AKV-G1-2,and sf9 cells were transfected with Cellfectin Reagent II to obtain recombinant proteins;and final-ly,the recombinant protein expression was detected and analyzed by Western blot.The results of the software analysis showed that the 189-397 aa peptide was a hydrophilic protein with a transmembrane region;and the protein had multiple potential phosphorylation sites,had rich potential epitopes,and belonged to the dominant antigen peptide.Next,the peptide was chosen for truncated eukaryotic expression,and Western blot identification was carried out using the AKV antibody-positive serum.The results showed that the recombinant protein could be specifically recognized,and the expected protein reaction band appeared,indicating that the G1-2 protein was successfully expressed with a molecular weight of about 27 kDa.This study laid a foundation for future development of diagnostic reagents for Akabane virus with trun-cated expression products as antigens.

关 键 词：赤羽病病毒 G1蛋白 杆状病毒表达 生物信息学 截短表达 

分 类 号：S855[农业科学—临床兽医学]