中国人兽共患病学报2026，Vol.42Issue(4)：348-355,8.DOI:10.3969/j.issn.1002-2694.2026.00.025
结核分枝杆菌B/R菌株灭活全菌裂解液的制备及其免疫原性与安全性评价
Preparation,immunogenicity,and safety of inactivated whole-cell lysate of the Mycobacterium tuberculosis B/R strain
摘要
Abstract
This study was aimed at developing a safe and effective inactivated whole-cell lysate of a novel Mycobacterium tubercu-losis(M.tb)fusion strain(B/R strain)by using ultrasonic microbubble cavitation combined with enzymatic lysis(lysozyme/glycine),hereafter referred to as the ultrasonically-killed B/R strain(UK-B/R).Inactivation efficacy,preservation of conformational epitopes,immunogenicity,and safety were systematically evaluated.The B/R strain was inactivated with ultrasonic microbubble cavitation(30 minutes)combined with pretreatment with lysozyme-glycine(6 hours).The inactivation was verified through colony culture,flow cyto-metric analysis of FDA esterase activity,and N01/PI fluorescence staining.The effects of the inactivation process on key M.tb anti-genic proteins(ESAT6,CFP10,MPT64,and Ag85B)were assessed with western blotting.C57BL/6J mice were subcutaneously im-munized and divided into four groups:PBS,BCG,B/R,and UK-B/R.At weeks 2 and 4 post-immunization,serum cytokines(IFN-γ,TNF-α,and IL-2)and antigen-specific antibodies were measured with ELISA.The cytotoxicity of UK-B/R toward RAW 264.7 macrophages was analyzed with CCK-8 assays.Culture colony assays,bacterial esterase activity tests,and fluorescence staining to-gether confirmed the complete inactivation of the B/R strain.Western blot analysis indicated that the B/R strain retained key tuberculo-sis antigens such as ESAT6/CFP10(11 kDa),MPT64(25 kDa),and Ag85B(37 kDa)both before and after inactivation.Subcutane-ous immunization with UK-B/R induced high levels of cytokines,whose expression showed time-dependent dynamics and sex-specific differences,and elicited high levels of antigen-specific IgG in serum.Furthermore,no adverse reactions were observed in mice immunized with UK-B/R.In vitro experiments demonstrated that various doses of UK-B/R had no effects on macrophage viabil-ity.This study demonstrated that UK-B/R,prepared by ultrasonic microbubble cavitation combined with enzymatic lysis,demon-strated complete inactivation,preserved critical mycobacterial antigens,effectively activated both cellular and humoral immune re-sponses,and exhibited a favorable safety profile.Therefore,this strain has potential as a candidate inactivated whole-cell vaccine against tuberculosis.关键词
新型结核杆菌融合菌株(B/R菌株)/灭活全菌疫苗/超声微气泡物理损伤技术/酶解法/免疫原性Key words
novel Mycobacterium tuberculosis fusion strain(B/R strain)/inactivated whole-cell vaccine/ultrasonic microbubble cavitation/enzymatic lysis/immunogenicity分类
医药卫生引用本文复制引用
盘智敏,章乐,张万江,徐梅,徐锋,李晓梅,董江涛,张杰,吴芳,梁粟,柳小玲..结核分枝杆菌B/R菌株灭活全菌裂解液的制备及其免疫原性与安全性评价[J].中国人兽共患病学报,2026,42(4):348-355,8.基金项目
2023年兵团重点领域科技攻关计划项目(No.2023AB053) （No.2023AB053）
国家自然科学基金资助项目(No.82360321) （No.82360321）
2022年兵团指导性科技计划项目(No.2022ZD043). Key Field Science and Technology Research and Development Program Project of the Xinjiang Production and Con-struction Corps(No.2023AB053) （No.2022ZD043）
National Natural Science Foundation of China(No.82360321) （No.82360321）
Corps Guiding Science and Tech-nology Plan Project(No.2022ZD043). （No.2022ZD043）
