摘要：
目的 构建周期型马来丝虫半胱氨酸蛋白酶抑制剂/3-磷酸甘油醛脱氢酶(BmCPI/BmGAPDH)复合基因重组质粒,并转染人宫颈癌细胞(Hela)进行重组蛋白表达.将重组质粒和相应表达蛋白进行免疫学研究比较,为研制新型的抗丝虫基因工程疫苗提供理论及实验依据.方法 扩增周期型马来丝虫目的编码基因.将目的基因片段和载体分别双酶切后进行连接,构建复合基因重组质粒pcDNA3.1(+)-BmCPI/BmGAPDH,鉴定后转染Hela细胞,以G418筛选转染细胞,进而通过RT-PCR和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定G418筛选后的单克隆抗性细胞株.亲和层析纯化所表达的重组蛋白并通过免疫蛋白印迹法(Western blot)对纯化的重组蛋白进行生物学鉴定.60只BALB/c小鼠分5组,每组12只,第2、4、6周分别免疫1次.磷酸盐缓冲液(PBS)对照组为注射PBS 100 μl;空质粒/CpG对照组为注射pcDNA3.1 100 μg和CpG 30 μg;复合重组质粒/CpG组为注射pcDNA3.1-BmCPI/BmGAPDH 100 μg和CpG 30 μg;复合重组蛋白/CpG组为注射pcDNA3.1-BmCPI/BmGAPDH复合重组蛋白50 μg和CpG 30 μg;复合重组质粒/复合重组蛋白/CpG组前2次注射pcDNA3.1-BmCPI/BmGAPDH 100 μg和CpG 30 μg;后1次注射pcDNA3.1-BmCPI/BmGAPDH复合重组蛋白50 μg和CpG 30 μg.用RT-PCR方法检测肌肉组织内目的基因;用酶联免疫吸附试验(ELISA)、特异性增殖试验(MTT法)分别检测免疫小鼠血清抗体效价、T淋巴细胞刺激增殖指数和血清中细胞因子干扰素(INF)-γ、白细胞介素(IL)-4水平.结果 复合重组质粒pcDNA3.1 (+)-BmCPI/BmGAPDH免疫小鼠后,从小鼠肌肉组织扩增出目的基因.复合重组质粒/复合重组蛋白/CpG组小鼠免疫4、6周后血清抗体效价(1 695.12±1.43、3 199.63±1.34)均高于复合重组质粒/CpG组(712.69±1.08、1 510.08±1.43,P均＜0.05)和复合重组蛋白/CpG组(800.02±1.34、1 510.08±1.43,P均＜0.05);第6周的复合重组质粒/复合重组蛋白/CpG组小鼠淋巴细胞刺激增殖指数(1.629±0.235)高于复合重组蛋白组(1.248±0.110,P＜0.05);免疫4、6周后,复合重组质粒/复合重组蛋白/CpG组和复合重组质粒/CpG组小鼠血清IFN-γ水平[(101.660±5.101)、(178.265±7.139)mg/L,(102.067±3.722)、(115.148±6.031)mg/L]均高于复合重组蛋白组[(75.438±2.102)、(82.004±3.777) mg/L,P均＜0.05];免疫后6周,复合重组质粒/复合重组蛋白/CpG组和复合重组蛋白/CpG组的小鼠血清IL-4水平[(75.385±3.318)、(46.363±3.672)mg/L]均明显高于复合重组质粒/CpG组[(36.691±3.443)mg/L,P均＜0.05).结论 pcDNA3.1-BmCPI/BmGAPDH核酸疫苗和相应蛋白疫苗均可诱导BALB/c小鼠产生特异性体液和细胞免疫应答反应.核酸疫苗-蛋白疫苗联合免疫效果有明显的优势.%Objective To construct a plasmid DNA vector expressing cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase of periodic Brugia malayi(BmCPI/BmGAPDH),and purify the recombinant protein after transfecting the vector into human cervical carcinoma cells(Hela) for expression.To make a comparison of immunity efficacy between the recombinant plasmid and the homologous protein and to a lay theoretic and experimental basis for developing novel anti-filarial genetic engineering vaccines.Methods The amplified genes BmCPI and BmGAPDH and a plasmid vector were double enzymes digested and ligated to construct a recombinant plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH,and this plasmid was transfected to Hela cells after being identified.G418 was used for screening transfectants,and the monoclonal resistant cell strain was determined by RT-PCR and SDS-PAGE.The recombinant protein was purified by affnity chromatography and identified by Western blotting.Sixty BALB/c mice were divided into 5 groups,12 per group,and they were immunized at 2,4,and 6 weeks.Mice in control groups were injected with PBS 100 μ1 or pcDNA3.1 100 μg/CpG 30 μg,and mice in experimental groups were injected with pcDNA3.1-BmCPI/BmGAPDH 100 μg/CpG 30 μg,the recombinant protein 50 μg/CpG 30 μg,or pcDNA3.1-BmCPI/BmGAPDH 100 μg/CpG 30 μg for the first two times and the recombinant protein 50 μg/CpG 30 μg for the third one.Target genes in muscular tissue were detected by RT-PCR.Stimulation index (SI) of the spleen lymphocytes of immunized mice was measured by specific proliferation test(MTT assay).Serum antibody titer and the level of secreted IL-4 and INF-γ in mice were detected by ELISA.Results The gene BmCPI/BmGAPDH was detected in the injected muscle of mice after injection with pcDNA3.1 (+)-BmCPI/BmGAPDH.Serum antibody titers of mice in plasmid/protein/CpG group (1 695.12 ± 1.43,3 199.63 ± 1.34) were higher than those in plasmi d/CpG group(712.69 ± 1.08,1 510.08 ± 1.43,P ＜ 0.05) and those in protein/CpG group (800.02 ± 1.34,1 510.08 ± 1.43,P ＜ 0.05) 4 and 6 weeks after immunization.The proliferation of spleen T lymphocytes seen on MTT assay was higher in plasmid/protein/CpG group(1.629 ± 0.235) than that in protein group (1.248 ± 0.110,P ＜ 0.05).The levels of serum INF-γ were higher in plasmid/protein/CpG group [(101.660 ± 5.101),(178.265 ± 7.139)mg/L] and plasmid/CpG group[(102.067 ± 3.722),(115.148 ± 6.031)mg/L] than those in protein/CpG group[(75.438 ± 2.102),(82.004 ± 3.777)mg/L,P ＜ 0.05] 4 and 6 weeks after immunization.The level of serum IL-4 from plasmid/protein/CpG group[(75.385 ± 3.318)mg/L] and protein/CpG group [(46.363 ± 3.672)mg/L] was significantly higher than that from plasmid/CpG group[(36.691 ± 3.443)mg/L,P＜ 0.05] 6 weeks after immunization.Conclusions Both the DNA vaccine pcDNA3.1 (+)-BmCPI/BmGAPDH and its homologous protein could elicit specific humoral and cellular immune responses in immunized BALB/c mice.Efficacy of DNA/protein vaccine has obvious advantages.
