中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
Chinese Journal of Microbiology and Immunology
2015年
9期
646-652
,共7页
李晓燕%郭丽茹%孔梅%邹明%苏旭
李曉燕%郭麗茹%孔梅%鄒明%囌旭
리효연%곽려여%공매%추명%소욱
人偏肺病毒%多表位抗原%免疫应答
人偏肺病毒%多錶位抗原%免疫應答
인편폐병독%다표위항원%면역응답
Human metapneumovirus%Multi-epitope antigen%Immune response
目的：评价本实验室构建的人偏肺病毒多表位抗原（ MEA ）的免疫应答水平。方法4～6周龄雌性SPF级BALB／c小鼠分为7组：MEA＋含CpG基序的寡聚脱氧核苷酸（ oligodeoxynucle-otides containing CpG motifs ，CpG ODN）腹腔注射组i．p．，MEA＋Alum腹腔注射组i．p．，MEA＋Alum＋CpG ODN腹腔注射组i．p．，MEA＋CpG ODN滴鼻组i．n．，MEA＋Alum＋CpG ODN滴鼻组i．n．，以上5组分别于0 d、14 d、21 d免疫3次；MEA＋Quickantibody5W肌肉注射组i．m．，分别于0 d、21 d免疫2次，同时设未处理对照组，末次免疫后2周杀鼠进行相应检测。采用ELISA法检测血清IgG、IgG1、IgG2a和IgA抗体，MTS法进行淋巴细胞增殖试验，LDH法检测杀伤性T淋巴细胞杀伤活性，流式细胞仪检测CD4＋／CD8＋T细胞，悬浮芯片法检测脾细胞分泌细胞因子。结果 MEA免疫后除滴鼻免疫组外，其他组均产生了高滴度IgG抗体，抗体效价均＞104。除滴鼻免疫组外，其余各组IgG1与IgG2a抗体均显著存在。3组免疫动物血清中检测到了IgA，其中MEA＋Alum＋CpG ODN i．p．组IgA效价最高为2．15×103。与对照组相比，MEA＋Alum i．p．、MEA＋Alum＋CpG ODN i．p．和 MEA＋Quickantibody5W i．m．淋巴细胞增殖指数有明显增高（P＜0．05）。腹腔免疫组及MEA＋Quickantibody5W 肌肉免疫组CTL杀伤活性均明显增强（P＜0．05）。 MEA＋CpG ODN i．p组Th1型细胞因子均分泌增多，其中IL-2和IFN-γ增幅最明显，3种Th2型细胞因子分泌均明显增多。 MEA＋Alum i．p．组IFN-γ稍有增加， IL-4、IL-5、IL-10均明显增加，MEA＋Alum＋CpG ODN i．p．组IFN-γ增幅最明显，IL-4、IL-5、IL-10均明显增加。 MEA＋Quickantibody5W i．m组IFN-γ增幅最明显，IL-5、IL-10明显增加，GM-CSF也明显增加。除滴鼻免疫组外，其余各组脾细胞中CD4＋／CD8＋T细胞稍有增加（P＜0．05）。结论 MEA在不同佐剂作用下可产生高滴度的特异性抗体，也可激活CTL。 CpG ODN可引起Th1／Th2平衡免疫应答，CpG ODN和铝镁佐剂合用免疫效果最好，商品化佐剂Quickantibody5W也可产生相似的效果。本研究为今后hMPV表位疫苗开发、诊断方法建立及hMPV流行病学研究等奠定了基础。
目的：評價本實驗室構建的人偏肺病毒多錶位抗原（ MEA ）的免疫應答水平。方法4～6週齡雌性SPF級BALB／c小鼠分為7組：MEA＋含CpG基序的寡聚脫氧覈苷痠（ oligodeoxynucle-otides containing CpG motifs ，CpG ODN）腹腔註射組i．p．，MEA＋Alum腹腔註射組i．p．，MEA＋Alum＋CpG ODN腹腔註射組i．p．，MEA＋CpG ODN滴鼻組i．n．，MEA＋Alum＋CpG ODN滴鼻組i．n．，以上5組分彆于0 d、14 d、21 d免疫3次；MEA＋Quickantibody5W肌肉註射組i．m．，分彆于0 d、21 d免疫2次，同時設未處理對照組，末次免疫後2週殺鼠進行相應檢測。採用ELISA法檢測血清IgG、IgG1、IgG2a和IgA抗體，MTS法進行淋巴細胞增殖試驗，LDH法檢測殺傷性T淋巴細胞殺傷活性，流式細胞儀檢測CD4＋／CD8＋T細胞，懸浮芯片法檢測脾細胞分泌細胞因子。結果 MEA免疫後除滴鼻免疫組外，其他組均產生瞭高滴度IgG抗體，抗體效價均＞104。除滴鼻免疫組外，其餘各組IgG1與IgG2a抗體均顯著存在。3組免疫動物血清中檢測到瞭IgA，其中MEA＋Alum＋CpG ODN i．p．組IgA效價最高為2．15×103。與對照組相比，MEA＋Alum i．p．、MEA＋Alum＋CpG ODN i．p．和 MEA＋Quickantibody5W i．m．淋巴細胞增殖指數有明顯增高（P＜0．05）。腹腔免疫組及MEA＋Quickantibody5W 肌肉免疫組CTL殺傷活性均明顯增彊（P＜0．05）。 MEA＋CpG ODN i．p組Th1型細胞因子均分泌增多，其中IL-2和IFN-γ增幅最明顯，3種Th2型細胞因子分泌均明顯增多。 MEA＋Alum i．p．組IFN-γ稍有增加， IL-4、IL-5、IL-10均明顯增加，MEA＋Alum＋CpG ODN i．p．組IFN-γ增幅最明顯，IL-4、IL-5、IL-10均明顯增加。 MEA＋Quickantibody5W i．m組IFN-γ增幅最明顯，IL-5、IL-10明顯增加，GM-CSF也明顯增加。除滴鼻免疫組外，其餘各組脾細胞中CD4＋／CD8＋T細胞稍有增加（P＜0．05）。結論 MEA在不同佐劑作用下可產生高滴度的特異性抗體，也可激活CTL。 CpG ODN可引起Th1／Th2平衡免疫應答，CpG ODN和鋁鎂佐劑閤用免疫效果最好，商品化佐劑Quickantibody5W也可產生相似的效果。本研究為今後hMPV錶位疫苗開髮、診斷方法建立及hMPV流行病學研究等奠定瞭基礎。
목적：평개본실험실구건적인편폐병독다표위항원（ MEA ）적면역응답수평。방법4～6주령자성SPF급BALB／c소서분위7조：MEA＋함CpG기서적과취탈양핵감산（ oligodeoxynucle-otides containing CpG motifs ，CpG ODN）복강주사조i．p．，MEA＋Alum복강주사조i．p．，MEA＋Alum＋CpG ODN복강주사조i．p．，MEA＋CpG ODN적비조i．n．，MEA＋Alum＋CpG ODN적비조i．n．，이상5조분별우0 d、14 d、21 d면역3차；MEA＋Quickantibody5W기육주사조i．m．，분별우0 d、21 d면역2차，동시설미처리대조조，말차면역후2주살서진행상응검측。채용ELISA법검측혈청IgG、IgG1、IgG2a화IgA항체，MTS법진행림파세포증식시험，LDH법검측살상성T림파세포살상활성，류식세포의검측CD4＋／CD8＋T세포，현부심편법검측비세포분비세포인자。결과 MEA면역후제적비면역조외，기타조균산생료고적도IgG항체，항체효개균＞104。제적비면역조외，기여각조IgG1여IgG2a항체균현저존재。3조면역동물혈청중검측도료IgA，기중MEA＋Alum＋CpG ODN i．p．조IgA효개최고위2．15×103。여대조조상비，MEA＋Alum i．p．、MEA＋Alum＋CpG ODN i．p．화 MEA＋Quickantibody5W i．m．림파세포증식지수유명현증고（P＜0．05）。복강면역조급MEA＋Quickantibody5W 기육면역조CTL살상활성균명현증강（P＜0．05）。 MEA＋CpG ODN i．p조Th1형세포인자균분비증다，기중IL-2화IFN-γ증폭최명현，3충Th2형세포인자분비균명현증다。 MEA＋Alum i．p．조IFN-γ초유증가， IL-4、IL-5、IL-10균명현증가，MEA＋Alum＋CpG ODN i．p．조IFN-γ증폭최명현，IL-4、IL-5、IL-10균명현증가。 MEA＋Quickantibody5W i．m조IFN-γ증폭최명현，IL-5、IL-10명현증가，GM-CSF야명현증가。제적비면역조외，기여각조비세포중CD4＋／CD8＋T세포초유증가（P＜0．05）。결론 MEA재불동좌제작용하가산생고적도적특이성항체，야가격활CTL。 CpG ODN가인기Th1／Th2평형면역응답，CpG ODN화려미좌제합용면역효과최호，상품화좌제Quickantibody5W야가산생상사적효과。본연구위금후hMPV표위역묘개발、진단방법건립급hMPV류행병학연구등전정료기출。
Objective To evaluate the immune response triggered by an in-house constructed hu-man metapneumovirus multi-epitope antigen ( MEA) in a mouse model .Methods Female SPF BALB/c mice at age 4-6 weeks were used in the study and divided into 7 groups.Mice in the five groups including MEA+oligodeoxynucleotides containing CpG motifs ( CpG ODN) intraperitoneal injection ( i.p.) treatment group, MEA+Alum i.p.treatment group, MEA+Alum+CpG ODN i.p.treatment group, MEA+CpG ODN intranasal (i.n.) treatment group and MEA+Alum+CpG ODN i.n.treatment group were immunized three times on days 0, 14 and 21, and those in the other experimental group were immunized intramuscularly with MEA+Quickantibody5W on days 0 and 21.A control group without treatment was set up accordingly .All mice were sacrificed two weeks after the last immunization .Antibodies including IgG , IgG1, IgG2a and IgA in serum samples were detected by ELISA .MTS assay was performed to analyze the proliferation of lympho-cytes.The cytotoxicity of cytotoxic T lymphocytes (CTL) was measured by LDH assay.Flow cytometry was used to detect T lymphocyte subsets .The cytokines secreted by T helper cells ( Th1 and Th2) were analyzed with Bio-Rad Liquid Chips.Results High titers of IgG, IgG1 and IgG2a antibodies were produced in MEA treated mice except for those in intranasal treatment groups .Serum samples from three groups including the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups were positive for IgA antibody .The highest titer of IgA antibody was detected in mice from the MEA+Alum+CpG ODN i.p.treatment group, which was 2.15×103.Compared with the control group, significantly enhanced proliferation of lymphocytes was observed in the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups (P<0.05).Enhanced cytotoxic activities of CTL were observed in mice with ip.and i.m.treatments as compared with those in control group (P<0.05).The levels of CD4+/CD8+T cells were slightly increased in mice from the MEA+CpG ODN i.p., MEA+Alum+CpG ODN i.p. and MEA+Quickantibody5W i.m.treatment groups as compared with those in control group (P<0.05).In-creased secretion of IL-2, IFN-γand Th2-type cytokines including IL-4, IL-5 and IL-10 were detected in mice from the MEA+CpG ODN i.p.treatment group.The MEA+Alum i.p.treated mice showed a slightly increased secretion of IFN-γand significantly increased secretions of IL-4, IL-5 and IL-10.Significantly in-creased secretions of IFN-γ, IL-4, IL-5 and IL-10 were detected in mice from the MEA+Alum+CpG ODN i.p.treatment group.Significantly increased secretions of IFN-γ, IL-5, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in mice from the MEA+Quickantibody5W i.m.treatment group.Conclusion MEA together with different adjuvants could stimulate high titers of specific antibodies , increase the proliferation of lymphocytes and enhance the cytotoxic activities of CTL .CpG ODN could bal-ance the Th1/Th2-mediated immune responses , and the balance could be enhanced when using CpG ODN in combination with Alum .A similar effect could be achieved by using the commercial adjuvant Quickanti -body5w.This study has paved the way for further investigation on the development of hMPV epitope vaccines and diagnostic reagents for hMPV as well as the epidemiological study of hMPV .
