多房棘球绦虫elp基因核酸疫苗诱生免疫应答的实验研究

Immune response in mice induced by nucleic acid vaccine wih elp gene of Echinococcus multilocularis

目的观察多房棘球绦虫elp基因核酸疫苗免疫BALB/c小鼠引起的体液和细胞免疫应答。方法30只BALB/c小鼠随机分成3组:Ⅰ组,肌注空质粒(pcDNA3.1)100μg/鼠;Ⅱ组,肌注多房棘球绦虫elp基因真核表达重组质粒(pcD-ELP)100μg/鼠;NS组,肌注等体积生理盐水。每2周免疫1次,共3次。ELISA方法检测免疫后不同时间产生的特异性IgG1,IgG2a和IgG 2 b水平。双抗体夹心ELISA法检测免疫鼠脾脏单个核细胞(PMNC)在受到特异抗原刺激后,产生IL-4I、L-12和IFN-γ的能力,3H-TdR掺入法检测脾淋巴细胞的增值能力。结果首次免疫后4周,Ⅱ组小鼠血清可检测到特异性IgG1I、gG2a和IgG2b抗体,随免疫时间和免疫次数的增加3种特异性抗体的OD值逐渐增高,实验结束时(首次免疫后7周)3种特异性抗体OD值均最高。Ⅰ组和Ⅱ组小鼠脾PMNC培养上清中IFN-γ浓度分别为50 pg/ml和55 pg/ml,受特异抗原刺激后分别为44 pg/ml和101.5 pg/ml。NS组IFN-γ及各组IL-4和IL-12均未检出。Ⅰ组和Ⅱ组小鼠脾淋巴细胞增殖能力增高,其淋巴细胞CPM值分别为NS组的85倍和123倍,受到特异抗原或刀豆素刺激其淋巴细胞增殖更明显。结论多房棘球绦虫elp基因核酸疫苗可刺激BALB/c小鼠同时产生很强的细胞免疫和体液免疫应答。

To observe the humoral and cellular immune responses in BALB/c mice induced by the nucleic acid vaccine with the full-length elp gene of Echinococcus multilocularis. 30 BALB/c mice were divided into 3 groups, and in Ⅰ , Ⅱ and NS gooups of mice, each mouse was intramuscularly iniected with 100μg of plasmid pcDNA3.1, 100μg of the eukaryotlc expression recombinant plasmid pcD-ELP with the full-length elp gene of Echinococcus multilocularis or equal volume of normal saline respectively. Each mouse was injected three times at a two-week interval. Before and at different times after immunization, specific antibodies of IgG1, IgG2a and IgG2b were detected with ELISA. IFN-γ, IL-4 and IL-12 produced by peripheral mononuclear eell（PMNC） of spleen defore and after stimulation of E. multilocularis antigen were examined with Double-Antibody- Sandwich EL1SA kits. The effects of the nucleic acid vaccine on the proliferation of spleen lymphocytes were evaluated by a ^3H-TdR method. In Ⅱ groups of mice, specific IgG1, IgG2a and IgG2b antibodies were detectable in four weeks after the first dose immunization, then gradually increased and reached highest OD value at the end of experiment, i.e. at the end of the seventh week after first immunization. The amount of IFN-γ produced by spleen PMNC of Ⅰ and Ⅱ group reached 50μg/ml and 55μg/ml in their culture supernatants respectively. After specific antigen stimulation, the amount of IFN-γ of Ⅰ and Ⅱ group were 44pg/ml and 101. 5pg/ml respectively. However, INF-γ in NS group, IL-4 and IL-12 in all 3 groups were below the threshold of the kits. Compared with that of NS group, the proliferations of spleen lymphocytes of both Ⅰ and Ⅱ group were enhanced. Their CPM values were 85 and 123 times as high as that of NS group. The capacities of lymphocyte proliferation were even higher after in vitro stimulation of specific antigen or ConA. It is evident from the above observations that the nucleic acid vaccine with the full-length elp gene of E. multilocularis is able to induce both cellular and humoral immune responses in BALB/c mice.