摘要
为评价羊口疮病毒(OrfV)B2L、F1L基因融合真核表达质粒pVAX1-B2L-F1L免疫小鼠诱导的体液和细胞免疫应答及IL-2对其免疫作用的影响。本研究将构建的pVAX1-B2L-F1L真核质粒转染MDBK细胞后,采用RT-PCR和间接免疫荧光试验(IFA)检测B2L-F1L融合基因在MDBK细胞中的表达;将pVAX1-B2L-F1L、pVAX1-B2L-F1L+pVAX1-IL-2、pVAX1空载体、生理盐水对照组KM系小鼠通过后腿肌肉注射的方式免疫,采用ELISA方法检测免疫小鼠血清中OrfV特异性抗体以及Th1型(IL-2、IFN-γ)、Th2型(IL-4、IL-6)细胞因子;MTT法检测小鼠脾淋巴细胞增殖反应。结果显示,pVAX1-B2L-F1L重组质粒能够在MDBK细胞中表达;pVAX1-B2L-F1L+pVAX1-IL-2联合免疫组小鼠血清抗体及IL-2和IFN-γ水平均显著高于pVAX1-B2L-F1L组;该联合免疫组小鼠血清IL-4、IL-6细胞因子水平与pVAX1-B2L-F1L组相比差异不显著(p>0.05);该联合免疫组小鼠的脾淋巴细胞增殖水平高于pVAX1-B2L-F1L组(p<0.05)。由此表明,pVAX1-B2L-F1L能够诱导小鼠产生OrfV特异性体液免疫和细胞免疫应答,联合pVAX1-IL-2诱导的免疫反应以细胞免疫应答为主,且能够促进Th1型细胞因子的分泌。本研究为OrfV基因工程疫苗的研制提供了参考依据。
The aim of this study was to evaluate the effect of OrfV F1L and B2L fusion gene eukaryotic expression plasmid pVAX1-B2L-F1L alone or combined with IL-2 on mice humoral and cellular immune responses. In this study, pVAX1-B2L-F1L eukaryotic plasmid was transfected into MDBK cells, and the expression of B2L-F1L fusion gene in MDBK cells was detected by RT-PCR and indirect immunofluorescence assay(IFA). The constructed pVAX1-B2L-F1L, pVAX1-B2L-F1L+pVAX1-IL-2, pVAX1 empty carrier and normal saline control KM-line mice were immunized by hind leg intramuscular injection of the mice. Specific OrfV serum antibody, Th1 type cytokines(IL-2, IFN-γ) and Th2 type cytokines(IL-4, IL-6) were detected by ELISA. MTT assay was used to detect lymphocyte proliferation in mice. The results showed that pVAX1-B2L-F1L recombinant plasmid was highly expressed in MDBK cells. The amount of anti-OrfV antibodies, cytokines of IL-2 and IFN-γ in pVAX1-B2L-F1L + pVAX1-IL-2 combined immunization group significantly higher than those in pVAX1-B2L-F1L group. There were no significant differences of IL-4 and IL-6 cytokine level in serum between pVAX1-B2L-F1L group and pVAX1-IL-2 group(p>0.05). The spleen lymphocyte proliferation level of pVAX1-B2L-F1L + pVAX1-IL-2 group was higher than that of pVAX1-B2L-F1L group(p<0.05). Therefore,pVAX1-B2L-F1L can highly induce OrfV-specific humoral and cellular immunities in mice when it was combined with pVAX1-IL-2. The immune response induced by pVAX1-IL-2 is mainly cellular immune response, promoting the secretion of Th1 cytokines. The rsults provide reference for the development of OrfV genetic engineering vaccine.
作者
鲜思美
张友
李婷
杨倩
饶体宇
吴伯梅
包涛涛
闵德省
包细明
张益
XIAN Si-mei;ZHANG You;LI Ting;YANG Qian;RAO Ti-yu;WU Bo-mei;BAO Tao-tao;MIN De-sheng;BAO Xi-ming;ZHANG Yi(College of Animal Science of Guizhou University,Guiyang 550025,China;Institude of Animal Disease Research of Guizhou,Guiyang 550025,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第2期168-174,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
贵州省科学技术基金项目[黔科合LH字(2014)7667]
贵州省科技计划项目黔科合支撑[2018]2264
贵州省科学技术基金项目黔科合基础[2019]1113号。
