流行性乙型脑炎病毒NS4B蛋白的原核表达及其结构分析

Prokaryotic Expression and Structural Analysis of NS4B Protein of Japanese Encephalitis Virus

为了解流行性乙型脑炎病毒NS4B蛋白的结构及功能,本研究构建了乙脑病毒P3株ns4b基因原核表达载体,在大肠杆菌BL21中诱导表达蛋白,并通过Western blot确定所表达蛋白为目的基因的产物。DNAStar软件分析其核苷酸和氨基酸序列特征,建立遗传进化树,利用生物信息学方法对其理化性质、蛋白结构等方面进行预测。结果显示,P3株ns4b基因全长765 bp,GC含量54.64%,包含21个酶切位点,与其他毒株的遗传距离为0.8,说明ns4b基因非常保守;编码255个氨基酸,预期大小为27 KDa,其中有20个强碱性氨基酸、15个强酸性氨基酸,等电点为9.24,为碱性蛋白,中性环境中带正电。生物信息分析表明NS4B蛋白无信号肽,有3个跨膜区域;无糖基化位点,磷酸化位点26个;亲水性良好;其蛋白二级结构有较多α-螺旋与无规则卷曲,中间较大的螺旋和两端的无规则卷曲构成其特殊三级结构。试验成功构建了原核表达载体pET42b-NS4B,在大肠杆菌中以包涵体的形式表达较大量蛋白,大小与预期一致。JEV-ns4b是该病毒较为关键的非结构蛋白基因,可通过原核表达系统高效表达蛋白,研究其蛋白功能,有助于进一步优化乙型脑炎疫苗。

In order to understand the structure and function of NS4B protein of Japanese encephalitis virus,the prokaryotic expres sion vector of ns4b gene of P3 strain of Japanese encephalitis virus was constructed,and the expression protein was induced in E.coli BL21.DNAStar software analyzed nucleotide and amino acid sequence characteristics,established genetic evolution tree,and predict ed its physical and chemical properties,protein structure and other aspects by using bioinformatics method.The results showed that the total length of NS4B gene of P3 strain was 765 bp,GC content was 54.64%,including 21 enzyme cutting sites,and the genetic distance with other strains was 0.8,indicating that ns4b gene was very conservative;it encoded 255 amino acids,expected size was 27 kDa,including 20 strong basic amino acids,15 strong acid amino acids,and the isoelectric point was 9.24,which was alkaline protein,with positive charge in neutral environment.Biological information analysis showed that NS4B protein had three transmem brane regions,26 phosphorylation sites,no glycosylation sites,and good hydrophilicity.The secondary structure of NS4B protein had moreα-helix and irregular curl,and the middle large helix and the two ends of the irregular curl formed its special tertiary struc ture.The prokaryotic expression vector pet42b-NS4B was successfully constructed and expressed in the form of inclusion body in E.coli BL21.Jev-ns4b is a key non structural protein gene of the virus.It can express the protein efficiently through prokaryotic expres sion system and study its protein function,which is helpful to further optimize the Japanese encephalitis vaccine.