山羊痘病毒南疆株P32基因的原核表达及其结构分析  

Prokaryotic expression and structural analysis of P32 gene of Goatpox virus Nanjiang strain

作　　者：宋书婷 焦海宏[1] 张孝忠[1] 李有文[1,2] SONG Shuting;JIAO Haihong;ZHANG Xiaozhong;LI Youwen(College of Animal Seienee,Tarim University,Aral 843300,China;Tarim Key Laboratory of Livestock Technology,Xingjiang Production and Construction Corps,Aral 843300,China)

机构地区：[1]塔里木大学动物科学学院,新疆阿拉尔843300 [2]新疆生产建设兵团塔里木畜牧科技重点实验室,新疆阿拉尔843300

出　　处：《黑龙江畜牧兽医》2019年第4期83-88,180,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31360616;31760741);塔里木畜牧科技兵团重点实验室开放课题项目(HS201307);塔里木大学研究生基金项目(TDGRI201604)

摘　　要：为了研究新疆南疆分离的山羊痘病毒P32基因的特征,试验通过PCR扩增得到P32基因及其膜外区P32_(1～282)基因和膜外亲水区P32_(20～270)基因,分别构建了表达载体(pET42b-P32、pET42b-P32_(1～282)和pET28a-P32_(20～270))并进行了原核表达,对其核苷酸序列的同源性、遗传进化关系及推测的氨基酸序列、蛋白质二级结构进行了预测分析。结果表明:山羊痘病毒南疆株P32基因发生了较大突变,相对对照在第100～105位增加了6个核苷酸,编码K、N两个氨基酸,全长达到975 bp,编码324个氨基酸,与Gansu 2009株相似。跨膜分析显示P32蛋白膜外区为1～282位氨基酸,跨膜区为283～305位氨基酸,膜内区为306～324位氨基酸。二级结构分析发现其分子两端呈明显的疏水结构,抗原性较弱,而中间区域有较强的亲水性和较强的抗原性。蛋白质表达发现其全长在大肠杆菌中几乎没有表达,其膜外区可以表达,但表达量较低,难以纯化。说明山羊痘病毒P32蛋白是一个典型的膜蛋白,全长较难表达纯化,南疆株P32蛋白也具有这样的特性。To study the characteristic of P32 gene of Goatpox virus separated from the Southem Xinjiang,PCR amplifying method was used to obtain P32 gene,its extracellular region P321-282 and its extracellular hydrophlhc region P3220-270.Expression vectors pETd2b-P32,pET42b- P321-282and pET28a-P3220-270 were constructed respectively and were expressed prokaryoticly.The predicted nucleotide sequence's homology and genetic relationship,and its deduced amino acid sequence and secondary protein structure were analyzed.The results showed that there was a big mutation in P32 gene of Goatpox virus Nanjiang strain.Compared with control sequence,added 6 nucleotldes were inserted at position 100-105 which encoded two amino acids K and N.The full gene length was 975 bp and it encoded 324 amino acids(AA),which was similar with Gansu2009 strain.Transmembrane analysis showed that extracellular region of P32 protein located at position 1-282 AA,and transmembrane region at position 283-305 AA,intramembrane region at position 306-324 AA.Secondary structure analysis showed that both ends of the molecule presented a clear hydrophobic structure and weak antigenicity,while the intermediate region was strongly hydrophilic and had strong antigenicity.Protein expression showed that the full-length protein had almost no expression in Escherichia coli,and extracellular region was expressed in the form of inclusion body but with low quantity and difficult purification.The results suggested that P32 protein of Goatpox virus was a typical membrane protein;the full-length protein had difficult expression and purification;and P32 protein of Nanjiang strain also had such features.

关 键 词：山羊痘病毒 南疆株 P32蛋白 原核表达 结构分析 

分 类 号：S852.654[农业科学—基础兽医学] Q78[农业科学—兽医学]