兔出血症病毒TP株VP60基因DNA免疫小鼠的实验研究  

Immune responses in mice induced by recombinant plasmid containing VP60 gene of rabbit hemorrhagic disease virus

作　　者：原冬伟[1] 刘家森[1] 郭东春[1] 司昌德[1] 姜骞[1] 林欢[1] 穆亚芳[1] 刘行波[1] 曲连东[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/实验动物中心,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2011年第8期653-657,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室开放基金(SKLVBD201002)

摘　　要：为评价兔出血症病毒(RHDV)VP60基因在小鼠体内诱导产生体液免疫和细胞免疫情况,本研究构建了RHDV VP60基因的真核表达质粒pcDNA-VP60,并将其免疫小鼠。利用ELISA方法检测特异性抗体和小鼠外周血细胞因子水平,MTT法和流式细胞术(FACS)检测小鼠外周血T淋巴细胞增殖情况和T淋巴细胞亚群的动态变化。抗体检测结果显示,重组质粒免疫组抗体水平在免疫后5周～6周达到峰值,而且与对照组比较差异显著(p<0.05);细胞因子检测结果显示,重组质粒免疫组血清中IFN-γ、IL-2、IL-4因子水平随免疫时间延长而升高,并且能够维持较高水平,与对照组比较差异显著(p<0.05);T淋巴细胞检测结果显示,重组质粒免疫组T淋巴细胞明显增殖,CD4+T淋巴细胞数量免疫后明显增高,与对照组比较差异显著(p<0.05);所有检测指标显示重组质粒免疫组和灭活苗免疫组比较均无显著差异(p>0.05)。以上结果表明pcDNA-VP60重组质粒可以诱导小鼠产生特异性体液免疫和细胞免疫,为研制预防兔病毒性出血症的候选DNA疫苗提供了实验依据。To evaluate the humoral immunity and cell-mediated immunity induced by rabbit hemorrhagic disease virus （RHDV） VP60 gene in the mice, the VP60 gene was amplified by PCR and cloned into pcDNA3.1 （＋）. The resultant recombinant plasmid pcDNA-VP60 was intramuscular injected to the mice. Specific antibodies and cytokines were detected by ELISA; T-lymphocyte in peripheral blood was detected by MTT method and FACS. The results showed that pcDNA-VP60 could induced high level humoral immunity, the antibody titers of immunization group was significantly higher （p〈0.05） than that of the control group; the concentrations of IFN-γ IL-2 and IL-4 were increased post immunization and keeped a high level; T lymphocyte proliferation and percentage of CD4＋ T lymphocyte increased in the peripheral blood of recombinant plasmid group, and was significantly higher than that of the control group （p〈0.05）. In conclusion, pcDNA-VP60 could induce high humoral immunity and cell-medicate immunity, the immune response induced by pcDNA-VP60 in mice was similar to the commercial inactive RHDV vaccine and the pcDNA-VP60 could be used as a candidate DNA vaccine against RHDV.

关 键 词：兔出血症病毒 T淋巴细胞 DNA疫苗 ELISA 

分 类 号：S855.3[农业科学—临床兽医学]