布鲁氏菌重组L7/L12蛋白对小鼠免疫保护效果的评价  

Evaluation of immune protective efficacy in mice induced by Brucella L7/L12 recombinant protein

作　　者：谢珊珊 王月丽[1,2] 邓肖玉 席静 于水发 马忠臣 王震 孟闯[3] 苗玉和 徐艺玫[5] 易继海 陈创夫[1,2] XIE Shan-shan;WANG Yue-li;DENG Xiao-yu;XI Jing;YU Shui-fa;MA Zhong-chen;WANG Zhen;MENG Chuang;MIAO Yu-he;XU Yi-mei;YI Ji-hai;CHEN Chuang-fu(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for Zoonotic Infectious Disease Prevention and Treatment,Shihezi 832000,China;Jiangsu Key Laboratory of Zoonoses,Yangzhou 225009,China;Fujian Biotechnology Co.,LTD.,Nanping 350000,China;Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention,Urumqi 830000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]人兽共患传染性疾病防治协同创新中心,新疆石河子832000 [3]江苏省人兽共患病学重点实验室,江苏扬州225009 [4]福建圣维生物科技有限公司,福建南平350000 [5]新疆维吾尔自治区疾病预防控制中心,新疆乌鲁木齐830000

出　　处：《中国预防兽医学报》2023年第1期72-78,87,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(U1803236、32002245);江苏省人兽共患病学重点实验室资助项目(R2104)。

摘　　要：为探究布鲁氏菌候选保护性抗原L7/L12的免疫原性及其对小鼠的免疫保护效果,本研究经PCR扩增羊种布鲁氏菌16M株L7/L12基因,构建重组质粒pET-22b-L7/L12,经双酶切鉴定正确后转化E.coli BL21(DE3)中,通过IPTG诱导表达并纯化后利用SDS-PAGE与western blot检测。结果显示,获得了16 ku的重组L7/L12蛋白(rL7/L12),且纯化效果较好。将rL7/L12以100μg/只腹腔注射免疫6周龄SPF BALB/c小鼠,14 d后以50μg/只加强免疫。首免后7 d、14 d、21 d、28 d、35 d、42 d及49 d分别对所有小鼠尾静脉采血,通过间接ELISA方法检测小鼠血清中的IgG抗体、其亚型(IgG1、IgG2a)抗体水平及二者比值;并利用ELISA方法检测首免后21 d和35 d两组小鼠血清中细胞因子IL-2、IL-10和TNF-α的分泌水平。结果显示,首免后14 d,小鼠血清中IgG抗体水平迅速升高,于首免后21 d达峰值并持续至首免后49 d(P<0.01),对照组小鼠则一直无抗体产生;首免后14 d,rL7/L12组小鼠血清中的IgG1抗体水平极显著高于IgG2a抗体水平(P<0.01),且IgG1/IgG2a大于1;但首免后42 d,rL7/L12组小鼠血清中IgG2a抗体水平极显著高于IgG1(P<0.01),IgG2a/IgG1比值大于1。表明在免疫前期,rL7/L12主要诱导小鼠的Th2型体液免疫反应,免疫后期主要诱导小鼠Th1型细胞免疫反应。首免后21 d和35 d,rL7/L12组小鼠IL-2和TNF-α含量极显著高于PBS对照组(P<0.01),首免后35 d,该组小鼠抑炎因子IL-10的含量极显著低于PBS对照组(P<0.01)。首免后21 d和35 d,分别剖杀每组小鼠,分离小鼠脾淋巴细胞,利用ELISpot法检测各组小鼠脾淋巴细胞中IFN-γ的分泌水平。结果显示,首免后21 d,rL7/L12组小鼠脾淋巴细胞IFN-γ的分泌水平有所升高,但与PBS对照组无显著差异(P>0.05);但免疫后35 d,rL7/L12组小鼠脾淋巴细胞IFN-γ分泌水平显著高于PBS对照组(P<0.05)。上述结果表明,rL7/L12免疫后期刺激小鼠机体产生Th1型细胞免疫反应,与抗体检测结果相符。首免后42 d�To investigate the immunogenicity of Brucella candidate protective antigen L7/L12 and its immune-protective efficacy in mice,the L7/L12 gene of Brucella abortus strain 16M was amplified by PCR,and the recombinant plasmid pET-22b-L7/L12 was constructed and identified by double digestion reaction followed by transformation into E.coli BL21(DE3).After induced by IPTG,the expression and purification was examined by SDS-PAGE and western blot.The results showed that 16ku of recombinant L7/L12 protein(rL7/L12)was produced and purified successfully.The 6-week-old SPF BALB/c mice was administered intraperitoneally with rL7/L12 at a dose of 100μg/each and then boosted with 50μg/each after 14 days.Blood samples were collected from the tail vein of both groups at 7,14,21,28,35,42 and 49 days after the first immunization.The levels of IgG antibodies,their subtypes(IgG1 and IgG2a)and their ratios in the sera of the two groups of mice were measured by indirect ELISA,and the levels of IL-2,IL-10 and TNF-αin the serum of the two groups of mice were detected by ELISA at 21 and 35 days after the first immunization.The results showed that the levels of IgG antibodies in both rL7/L12 immunized groups increased rapidly 14 days after the first immunization,peaked at 21 days and continued to 49 days after the first immunization(P<0.01),while no antibody was produced in the control group.The IgG1 antibody level in the serum of rL7/L12 group was significantly higher than that of IgG2a antibody level at 14 days after the first immunization(P<0.01),and the IgG1/IgG2a ratio was greater than 1.However,42 days after the first immunization,the serum IgG2a antibody level in the rL7/L12 group was significantly higher than that of IgG1(P<0.01),and the IgG2a/IgG1 ratio was greater than 1,indicating that rL7/L12 mainly induced Th2 type(humoral immune response)in mice in the early stage of immunization,and mainly induced Th1 type(cellular immune response)in the late stage of immunization.The levels of IL-2 and TNF-αin the immunization group were

关 键 词：布鲁氏菌 保护效果 ELISPOT 

分 类 号：S852.61[农业科学—基础兽医学]