摘要
目的:研究鉴定激活mfgl2凝血酶原酶基因之冠状病毒3型或A59型鼠肝炎病毒(MHV-3,MHV-A59)核心(N)蛋白的功能区域. 方法:应用定点突变技术、与mfg12启动子共转染实验明确mfgl2凝血酶原酶基因之MHV-3或MHV-A59 N蛋白的功能区域.N蛋白内含I基因突变病毒株A1b 110和其野生株A1b 111体外感染Balb/cJ小鼠巨噬细胞、I基因表达载体与mfgl2启动子共转染实验阐明I蛋白在mfgl2基因激活中的作用. 结果:N蛋白包含由两个可变问隔区(A,B)隔开的三个结构区(Ⅰ,Ⅱ,Ⅲ),MHV-A59N蛋白I区可增强mfgl2转录活性,当其基因序列突变为非嗜肝性MHV-JHM或MHV- 21区序列时,则丧失激活mfgl2启动子转录活性的功能.I 基因突变病毒株Alb 110和其野生株Alb 111体外感染Balb/cJ小鼠巨噬细胞后对mfgl2的激活无显著差异,共转染实验阐明I蛋白并非mfgl2基因激活中的必备因素,该组mfgl2启动子转录活性与对照组无显著差异,而N蛋白可激活mfgl2启动子,使其转录活性提高62倍. 结论:鼠肝炎病毒N蛋白I区为激活mfgl2凝血酶原酶基因的病毒蛋白功能区域.
AIM: To investigate the responsible domain(s) of N protein and the I gene within the N gene of MHV-3 or MHV-A59 in the activation of mfgl2. METHODS: To investigate the responsible domain(s) of N protein of MHV-3 or MHV-A59 in the activation of fgl2 gene, four ways comparison of the N protein was carried out and the site directed mutated N gene expression constructs within domain I and domain Ⅲ were cotransfected respectively with mfgl2 promoter/luciferase reporter gene in CHO cells. Macrophages from Balb/cJ mice were infected with I gene mutated MHV virus Alb110 and its isogenic Alblll for 8-10 hours, procoagulant activity (PCA) were measured. MHV-A59 I gene expression construct was cotransfected with mfgl2 promoter-reporter gene in Chinese hamster ovary (CHO) cells, and luciferase activity was detected for the assessment of promoter function. RESULTS: Mutations of residues Gly-12, Pro-38, Asn-40, Gln-41 and Asn42 within domain I of the N protein of MHV-A59 to their corresponding residues were found in MHV-2 abrogated mfgl2 transcription, whereas mutation of other N protein domain Ⅲ did not affect mfgl2 gene transcription. Alb 110 and Alb 111 infected macrophages showed a remarkable increasing in PCA activity compared with no virus or MHV-2 or MHV-JHM infected macrophages. There was no significant difference in PCA activity between Alb 110, Alb 111 infected group and MHV-A59 group. Cotransfection I gene expression construct with a reporter construct containing mfgl2 promoter in CHO cells displayed no significant difference in luciferase activity compared with nontransfected CHO cells. CONCLUSION: Domain I of nucleocapsid protein of murine hepatitis virus strain 3 upregulates the transcription of mfgl2 prothrimbinase/fibroleukin gene. The MHV-A59 I gene is not essential for activation of mfgl2 gene. Our study may shed lights on the investigation of current worldwide-distributed disease, severe acute respiratory syndrome (SARS).
出处
《世界华人消化杂志》
CAS
2004年第3期594-599,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No NSFC30170846杰出青年科学基金项目
No.NSFC30225040
NSFC30125019首批教育部防治非典科技攻关项目
No.[2003]64973计划"SARS防治基础研究"
No.2003CB514100~~
