弓形虫抗原与霍乱毒素A2/B亚基复合基因在小鼠体内诱导的免疫反应

Induction of protective immunity by DNA vaccination in mice by DNA vaccinationwith a eukaryotic expression plasmid encoding the compound gene of Toxoplasma gondii antigen and the A2/B subunit of cholera toxin

目的 观察弓形虫主要表面抗原SAG1、SAG2 与霍乱毒素A2 /B亚基复合基因真核质粒经肌肉免疫小鼠所诱导的免疫反应。方法 将SAG1基因、SAG2 基因及CTXA2 /B基因定向连接插入真核表达质粒pcDNA3.1,经酶切及测序,获得pcDNA3.1 SAG1 SAG2 及pcDNA3.1 SAG1 SAG2 CTXA2 /B的重组子;碱裂解法大量制备经肌肉注射免疫BALB/c鼠,每只鼠经后腿肌肉注射质粒10 0 μg ,每2周免疫1次,共3次,以PcDNA3.1空质粒注射组及PBS组为对照,分别于每次免疫前断尾取血和免疫后4周取小鼠脾脏测定T淋巴细胞增殖活性及NK细胞活性,ELISA法测定IgG抗体。结果 免疫组小鼠的IgG抗体水平明显提高,NK细胞杀伤活性和T细胞增殖活性也明显增强。免疫鼠抗攻击感染的时间延长。结论 含有霍乱毒素的复合基因免疫小鼠后体液免疫和细胞免疫水平均有提高。

To assess the protective immunity in BALB/c mice immunized intramuscularly with eukaryotic expression plasmid encoding the compound gene of Toxoplasma gondii surface antigen SAG 1 and SAG 2 with or without cholera toxin CTXA 2/B, gene fragment encoding SAG1 and SAG 2 were amplified from the genomic DNA of T.gondii RH strain and CTXA 2/B subunit from recombinant plasmid pUABO24-CTXA 2/B by PCR. These genes were inserted into PCDNA3.1(+) eukaryotic expression vector by digestion with restrictive enzymes and by ligating reaction. Large scale preparation of these genes was possible by method of alkaline lysis and could be used for the intramuscular immunization of mice, in which BALB/c mice were injected with plasmid pcDNA3.1-SAG 1-SAG 2 and pcDNA3.1-SAG 1-SAG 2-CTXA 2/B at a dosage of 100 μg. Booster immunization was performed at 2 weeks intervals for 3 times. Control groups were injected with empty plasmid pcDNA3.1 and PBS. Before and 4 weeks after immunization, mouse spleens of immunized and control mice were taken to test the proliferative responses of T lymphocytes and the activities of NK cells by means of MTT assay, ELISA was used to detect the level of IgG antibodies produced. The experimental results showed that significant elevation of the specific IgG antibody level was observed in immunized mice, and the proliferative response of T lymphocytes as well as the activities of NK cells were highly enhanced in the mice immunized with pcDNA3.1-SAG 1-SAG 2-CTXA 2/B. The time of death of mice after challenge the vaccinated mice with tachyzoites of the T.gondii RH strain was prolonged in comparison with that of the control mice. It is concluded that this vaccine with compound genes can enhance both humoral and cell-mediated immune responses in the vaccinated BALB/c mice, and CTXA 2/B can act as a gene adjuvant to increase the immunogenicity of DNA vaccine.The design and development of multi-component DNA vaccine may be a helpful direction in the research of T.gondii vaccine.