传染性造血组织坏死病毒核衣壳蛋白的原核表达及抗原性分析

Prokaryotic expression and antigenicity analysis of the nucleocapsid gene of infectious hematopoietic necrosis virus

应用RT-PCR方法扩增了长度为1176 bp的IHNV-ZYX株编码核衣壳(N)蛋白基因,将N基因克隆至原核表达载体pET30b,并在大肠杆菌Rosetta(DE3)中得到了表达。通过SDS-PAGE分析表明,重组菌诱导后得到了预期大小约48 KD的N蛋白,与理论值相符;提取N蛋白的包涵体,并制备抗血清。间接ELISA和Western-blot-ting实验结果说明,表达的N蛋白与天然的IHNV N蛋白一样具有相同的抗原性。本试验结果为进一步研究分离株IHNV-ZYX N基因的免疫功能,建立灵敏高效的检测传染性造血组织坏死病的方法和研制基因工程疫苗奠定了重要的物质基础。

The gene encoding the viral nucleocapsid （N） was amplified by RT- PCR from IHNV-ZYX strain and cloned into pET30b vector. The expression of recombinant plasmid pET30b-N in E. coli Rosetta（DE3 ） was induced and detected by SDS-PAGE analysis. The molecular weight of expressed recombinant N protein was approximately 48 KD as predicted. The inclusion body containing fusion protein was extracted and immunized in rabbits to obtain the antisera against N protein. Antigenicity of N protein was analyzed by indirect ELISA and Westernblotting. The results showed that the expressed N protein has immunogenical and antigenical characters as native IHNV N protein. This study has laid an important material foundation for further studying the N gene function of IHNV-ZYX in immune protection, establishing sensitive and efficient methods in detecting infectious hematopoietic necrosis, and manufacturing genetic engineering vaccine.