禽冠状病毒IBV反向遗传株S基因定点突变、受体筛选与细胞适应

禽传染性支气管炎病毒（IBV）是冠状病毒的代表种，是目前已知RNA病毒中基因组最大的病毒之一，但H120，M41等疫苗株不能够在传代细胞上生长，其机理尚不清楚，针对该科学问题，申请者在成功构建IBV反向遗传株（R-H120，R-SAIBK，R-M41）基础上(自然科学基金30972201)，拟开展以下三方面研究：1、用反向遗传操作技术对IBV纤突蛋白S（38，43等）关键氨基酸位点进行定点突变，验证、筛选适应细胞的IBV毒株。2、采用酵母双杂交、免疫共沉淀、荧光共聚焦等技术，筛选IBV的细胞受体，将受体转染传代细胞，获得IBV可生长的传代细胞株。3、分段克隆验证S蛋白与受体结合位点，用生物信息学分析S蛋白与受体空间结构，阐明IBV适应细胞的分子机理。本项目的实施，可获得IBV细胞适应株，探明IBV细胞适应性机理，具有重要的科学价值和应用前景。

As one of the largest in RNA viruses, Infectious bronchitis virus (IBV) is a typical species of coronavirus. However, the mechanism of IBV vaccine strains (H120, M41) that isn't able to proliferate in passage cell is not understood. Aims to this objective, Based on constructed IBV reverse genetic system(R-H120, R-SAIBK and R-M41) (30972201),this research were done as follows: 1.For the purpose of verifying mutation sites of spike protein of IBV, reverse genetics technique is used to mutate several amino acid sites (38,43 etc.) of spike gene,and IBV strain that can cultured on cell were obtained.2.The yeast two-hybrid, co-immunoprecipitation and fluorescent confocal technologies are used to screen and verify IBV receptor, the verified receptor or receptor gene is transfected to passage cell to construct IBV adaptation cells. 3. By using subsection cloning technology, the receptor binding site will be verified in S protein, and some bioinformation analysis methods will be used to analyse the relationship between S protein and receptor in space structures. This research is expected to demonstrate IBV cellular adaptation mechanism, and it has critical scientific value and application prospect.