结核分枝杆菌Rv3425-16kD融合蛋白原核表达及其刺激ATB患者IFN-γ释放水平研究  

Prokaryotic expression of Rv3425-16kD fusion protein and its study on stimulation production of IFN-γin patients with active tuberculosis

作　　者：史阅韩 张雪莲[2] 解宝江 贾晓炜 SHI Yuehan;ZHANG Xuelian;XIE Baojiang;JIA Xiaowei(Cadre Diagnosis and Treat Section,the Eighth Medical Center of General Hospital of PLA,Beijing 100091,China;Beijing International Studies University Hospital,Beijing 100024,China)

机构地区：[1]解放军总医院第八医学中心干部诊疗科,北京100091 [2]北京第二外国语学院门诊部,北京100024

出　　处：《临床肺科杂志》2024年第6期915-920,共6页Journal of Clinical Pulmonary Medicine

基　　金：国家科技重大专项(No.2017ZX10201301-007)。

摘　　要：目的 原核系统表达与纯化结核分枝杆菌(mycobacterium tuberculosis, MTB)融合蛋白抗原Rv3425-16kD,检测其能否被活动性结核病(active tuberculosis, ATB)中肺结核患者和结核潜伏性感染(latent TB infection, LTBI)者外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)识别,探讨作为诊断抗原鉴别活动性结核感染的可行性。方法 利用原核表达系统表达融合蛋白Rv3425-16kD并进行亲和纯化,以Western Blot法鉴定;收集结核病医学部收治的的ATB患者、LTBI者和健康人外周血,分离PBMCs,分别用Rv3425-16kD融合蛋白、早期分泌靶抗原6/培养液蛋白10(ESAT-6/CFP10)和MTB全菌裂解液刺激后,提取细胞总RNA,荧光定量PCR方法检测γ-干扰素(Interferon-γ,INF-γ) mRNA的表达,通过t检验分析刺激前后PBMCs IFN-γ mRNA的表达差异。结果 原核系统表达及纯化的Rv3425-16kD融合蛋白相对分子质量均为37kD。健康人PBMCs刺激前,IFN-γ mRNA表达量为4.2±1.6,MTB全菌裂解液刺激后为14.7±3.5,差异具有统计学意义(P<0.001),另外两组刺激没有明显变化;ATB患者PBMCs刺激前,IFN-γ mRNA表达量为3.7±1.2,经Rv3425-16kD融合蛋白、ESAT-6/CFP10抗原多肽和H37Rv全菌裂解液分别刺激后IFN-γ mRNA表达量为:166.0±29.7、13.7±5.8和213.0±33.5,三组抗原在刺激前后IFN-γ mRNA表达量差异均具有统计学意义(P<0.001)。结论 原核重组的Rv3425-16kD融合蛋白可被ATB患者PBMCs细胞识别,具有较强抗原刺激作用和特异性,可用作MTB感染的预防及诊断。Objective To express and purify the Rv3425-16kD fusion protein antigen of Mycobacterium tuberculosis in prokaryotic system,and to test whether it can be recognized by PBMCs cells from patients with active tuberculosis or latent TB infection,and to explore the feasibility of Rv3425-16kD as diagnostic antigen for differentiating active tuberculosis infection.Methods The fusion protein Rv3425-16kD was expressed and purified through a prokaryotic system,and identified by Western blot.The peripheral blood of ATB patients,LTBI and healthy people were recruited from the tuberculosis medical department.PBMCs were isolated from peripheral blood and stimulated with Rv3425-16kD,whole bacterial Lysate of H37Rv strain and peptide Pool of ESAT-6/CFP10.Fluorescence quantitative PCR was used to detect the mRNA expression of IFN-γ,and the difference of IFN-γmRNA between unstimulated and stimulated PBMCs were analyzed by paired t test statistic method.Results The relative molecular weight of Rv3425-16kD fusion protein expressed and purified by prokaryotic system was 37kD.Before PBMCs stimulation,IFN-γmRNA expression was 4.2±1.6 in healthy subjects,and 14.7±3.5 after MTB lysate stimulation,and the difference was statistically significant(P<0.001).There was no significant change in the other two groups.Before PBMCs stimulation,IFN-γmRNA expression in ATB patients was 3.7±1.2.After stimulation with Rv3425-16kD fusion protein,ESAT-6/CFP10 antigen polypeptide and H37Rv whole bacterial lysate,respectively,the expression of IFN-γmRNA was as follows:166.0±29.7,13.7±5.8 and 213.0±33.5.There were significant differences in IFN-γmRNA expression among the three groups before and after stimulation(P<0.001).Conclusion The recombinant Rv3425-16kD fusion protein with strong antigenic stimulation and specificity can be recognized by PBMCs cells from ATB patients,and it can be used for the prevention and diagnosis to MTB infection.

关 键 词：结核分枝杆菌 Rv3425-16kD融合蛋白 外周血单个核细胞 Γ-干扰素 

分 类 号：R52[医药卫生—内科学]