周期型马来丝虫复合基因重组质粒和相应表达蛋白的免疫学研究  

Immunology comparision of composite gene recombinant plasmid and expressed protein of periodical Brugia malayi

作　　者：王慧[1] 方政[1] 徐倩[1] 陆施娟[1] 钱一言 徐怿琳 方浩[1] 徐邦生[1] 

机构地区：[1]南通大学医学院寄生虫学教研室,226001

出　　处：《中华地方病学杂志》2014年第3期280-285,共6页Chinese Journal of Endemiology

基　　金：南通市应用研究计划（BK2012017）;江苏省高等学校大学生实践创新训练计划（2012JSSPITP1571）

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（Westernblot）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PBS100μl；空质粒／CpG对照组为注射pcDNA3．1100μg和CpG30μg；复合重组质粒／CpG组为注射pcDNA3．1-BmCPI／BmGAPDH100μg和CpG30μ；复合重组蛋白／CpG组为注射pcDNA3．1-BmCPI／BmGAPDH复合重组蛋白50P-g和CpG30μg；复合重组质粒／复合重组蛋白／CpG组前2次注射pcDNA3．1-BmCPI／BmGAPDH100μg和CpG30μg；后1次注射pcDNA3．1-BmCPI／BmGAPDH复合重组蛋白50μ和CpG30μg。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（1695．12±1．43、3199．63±1．34）均高于复合重组质粒／CpG组（712．69±1．08、1510．08±1．43，P均〈0．05Objective 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 andexperimental 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 μl 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 （MTr 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 plasmid/CpG group（712.69 ± 1.08, 1 510.08 ± 1.43, P 〈

关 键 词：马来丝虫 半胱氨酸蛋白酶抑制剂 3-磷酸甘油醛脱氢酶 复合基因 表达蛋白 免疫 

分 类 号：R382[医药卫生—医学寄生虫学]