弓形虫ROP16I/IIIDNA疫苗不能诱导小鼠有效的保护性免疫

目的利用ROP16_I/III基因真核表达重组质粒免疫BALB/c雌性小鼠,检测小鼠体液免疫和细胞免疫,评估ROP16_I/III作为潜在的分子疫苗的价值。方法克隆Chinese 1型弓形虫ROP16_I/III, 将其插入真核表达载体pEGFP-C2中构建重组质粒pEGFP-ROP16_I/III,脂质体法转染T293细胞并观察体外表达。Western blotting分析鉴定。将36只SPF级小鼠随机均分为:①PBS组;②空质粒组;③pEGFP-ROP16 _I/III组。每2周肌注免疫1次(100 μg/只),共3次;于免疫前及每次肌注前1 d和末次免疫后2周收集小鼠血清,间接ELISA法检测血清抗弓形虫IgG。于末次免疫2周后取小鼠脾脏,分离淋巴细胞培养测细胞因子。剩余小鼠腹腔注射Chinese 1型弓形虫 Wh3株速殖子1 000个/只,观察小鼠攻击感染后的存活时间和存活率。结果真核表达载体构建成功,体外见到重组质粒pEGFP-ROP16_I/III在T293细胞的表达;pEGFP-ROP16_I/III末次免疫后血清中IgG水平及脾淋巴细胞细胞因子均无明显升高(P>0.05);攻击感染后,免疫组小鼠比对照组的存活时间无延长(P>0.05);生物信息学分析发现ROP16_I/III缺乏线性B细胞表位。结论Chinese1 型弓形虫的ROP16_I/III由于其分泌的定位和结构特点,不能诱导小鼠产生有效的抗Wh3株攻击感染的免疫保护。

Recombinant eukaryotic plasmid of ROP16I/III failed to provide efficient protection against toxoplasmosis in BALB/c mice

To construct the recombinant eukaryotic expression plasmids of Toxoplasma ROP16_I/III, and to assess the immune protection in BALB/c mice induced by ROP16_I/III DNA vaccine, T. gondii ROP16_I/III coding gene obtained by PCR amplification was inserted into the eukaryotic expression vector pEGFP-C2 to generate recombinant pEGFP-ROP16_I/III. T293 cells were transfected by Liposome method in vitro and the fusion protein expression was identified by Western blotting. Thirty-six mice were randomly divided into 3 groups with 12 in each: PBS control group; empty plasmid group; and pEGFP-ROP16_I/III group, respectively. Immunization was completed in each mouse by intramuscular injection once 2 weeks for 3 times. Sera at each time point were collected for detection of antibodies against Toxoplasma by indirect ELISA. After two weeks of the last immunization, mouse splenocytes were harvested and cultured for tests of cytokines in the supernatant. The remaining mice were injected with 1 000 Wh3 tachyzoites for each, followed by observation of survival time and mortality of the animals. The recombinant eukaryotic expression vector of pEGFP-ROP16_I/III was successfully generated and efficient expressions were noted both in the transfected T293 cells and in the muscles in which the plasmid vaccine was given. All vaccinated mice, however, did not present the increased IgG antibody titers and cytokine levels after pEGFP-ROP16_I/III immunization. Correspondingly, no difference was seen of either survival or mortality of the animals compared with the control. Our conclusion is that Toxoplasma ROP16_I/III DNA vaccine failed to provide BALB/c mice with efficient immune protection against virulent tachyzoites challenge due to its inaccessibility to host immune components and its lack of linear B epitope.