传染性皮下及造血组织坏死病毒NJ株ORF3基因的原核表达及生物信息学分析

The prokaryotic expression for ORF3 gene of infections hypodermal and hematopoietic necrosis virus NJ strain and its bioinformatics analysis

为探究本实验室分离的传染性皮下及造血组织坏死病毒NJ株(IHHNV-NJ)ORF3基因编码蛋白的结构特征,本实验根据ORF3基因序列设计引物,利用PCR方法克隆ORF3基因序列,并构建至原核表达载体p ET-32a(+)中。对成功构建的p ET32a-ORF3重组表达载体进行原核表达,获得49 ku的融合蛋白,符合预期大小。通过生物信息学软件对ORF3基因编码蛋白序列进行分析,结果显示,ORF3基因序列长度为990 bp,编码329个氨基酸;ORF3基因编码蛋白理论分子质量为37 385.2 u,等电点为7.22,为亲水性蛋白;该编码蛋白序列不存在跨膜区、信号肽切割位点;二级结构含有55.9%的α-螺旋、52.0%的β-折叠以及13.4%的β-转角;抗原表位分布较广泛,抗原性强;该编码蛋白序列不存在潜在的N-糖基化位点,存在13个潜在的O-糖基化位点和17个潜在的磷酸化位点。进化树结果表明,NJ株的ORF3基因编码蛋白序列与6株IHHNV序列同源性均高于96%,与厄瓜多尔株同源性最高,为99.7%。研究表明,ORF3基因编码IHHNV衣壳蛋白;O-糖基化位点可参与衣壳蛋白的组装过程及细胞侵染过程,磷酸化位点可参与病毒在对虾细胞内的增殖过程;ORF3编码蛋白序列保守性强,不影响病毒毒力和个体间感染能力。

To research the and hematopoietic necrosis characteristics and structural features of ORF3 gene from infections hypodermal virus NJ strain（IHHNV-NJ） isolated in the laboratory,the primers were designed upon ORF3 gene sequence. Then we used PCR method to clone the ORF3 gene sequence and inserted it into the prokaryotic expression vector pET-32a （ ＋ ）. The successfully constructed vector pET32a-ORF3 was transformed into BL21 （DE3） competent cells for prokaryotic expression. ORF3 gene sequence was studied by means of bioinformatics software. As the results showed,the length of ORF3 gene sequence was 990 bp, encoding 329 amino acids. The theoretical molecular mass of the protein was 37 385.2 u and the isoelectric point was 7.22. The grand average hydrophobicity was -0. 619 and the hydrophilic zone（ score 〈 0）was evenly and widely distributed, which revealed a good hydrophile. The ORF3 sequence did not have transmembrane regions and signal peptide. The secondary structure of the protein contained 55.9% of α-helix,52.0% of β-fold and 13.4% of B-corner. Epitopes were located in multiple sequence intervals, and the protein showed a strong antigenicity. There were 13 potential O-glycosylation sites and 17 potential phosphorylation sites, but with no potential N-glycosylation sites. Phylogenetic analysis showed that the highest homology was between NJ strain and Ecuador strain. Comparing NJ strain with Ecuador strain, Hawaii strain, Taiwan strain, Fujian strain, California strain and Thai strain, the homology was 99. 7%, 99.1% ,99.1% ,98.8% ,98.2% and 96.6%. The recombinant expression vector pET32a-ORF3 successfully expressed a 49 ku fusion protein, which conformed to the expected size. Research shows, ORF3 gene may encode IHHNV capsid protein. O-glycosylation sites can participate in the assembly of capsid proteins and cellular infection process. Phosphorylation sites can participate in the reproduction process of the virus inside the shrimp＇ s cells. ORF3 gene sequence has a strong conservative property that won＇ t impact the viral toxicity and infection ability among individuals. The study provided the guidance and laid the foundation for those researches such as the IHHNV ORF3 gene＇ s genetic information,gene functions and establishment of IHHNV molecular epidemiology methods.