摘要
目的：构建编码恶性疟原虫复合多价保护性抗原的基因的重组质粒，为进行基因免疫提供条件。方法：设计一对特异引物Ｐ１与Ｐ２，采用ＰＣＲ法扩增获取ＭＳＰ１中Ｃ端１９肽基因，纯化后用ＳａｌⅠ＋ＸｂａⅠ双酶切，把含复合基因ＰｆＣＭＲ的重组质粒ｐＷＲ４５０－１／ＰｆＣＭＲ用ＥｃｏＲⅠ＋ＳａｌⅠ双酶切，回收复合基因ＰｆＣＭＲ，将ＭＳＰ１１９基因与ＰｆＣＭＲ基因串联后与经ＥｃｏＲⅠ＋ＸｂａⅠ双酶切的真核表达质粒ｐｃＤＮＡ３进行重组，转化大肠杆菌ＪＭ１０９，经电泳初筛、双酶切鉴定及ＰＣＲ鉴定。结果：ＰＣＲ扩增获得３６３ｂｐ的ＭＳＰ１１９基因，重组克隆经双酶切鉴定及ＰＣＲ鉴定后获得正确重组克隆子ｐｃＤＮＡ３－ＰｆＣＭＲ－ＭＳＰ１１９（命名为ｐｃＤＮＡ３－Ｐｆ８），Ｐｆ８基因长度为６１８ｂｐ。结论：成功构建编码恶性疟原虫多价保护性抗原的基因的重组质粒ｐｃＤＮＡ３－Ｐｆ８。
AIM∶ To construct a recombinant plasmid DNA encoding multiantigens of Plasmodium falciparum and to provide the requirements for DNA immunization. METHODS:Two oligonucleotide primers were designed to amplify MSP1 19 , the purified PCR products were digested by SalⅠ+XbaⅠ, and the recombinant plasmid pWR450 1/PfCMR was digested by EcoRⅠ+SalⅠ to recover PfCMR gene . PfCMR and MSP1 19 gene fragments were linked and recombined with mammalian expression vector pcDNA3. RESULTS:The MSP1 19 gene fragment with about 363 base pairs were specifically amplified by using PCR technique. The positive recombinant pcDNA3 PfCMR MSP1 19 (named pcDNA3 Pf8) was screened and identified by agarose gel electrophoresis,endonulease digestion and PCR technique, the whole length of Pf8 is 618 bp . CONCLUSION:By specifically amplifying MSP1 19 gene at the C terminal of MSP1,a recombinant plasmid pcDNA3 Pf8 encoding multiantigens of Plasmodium falciparum was successfully constructed.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
1999年第1期12-15,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家"九五"攻关项目基金
广东省自然科学资助基金
国家教育部博士点基金
关键词
恶性疟原虫
PCR
核酸疫苗
克隆
MSA1
Plasmodium falciparum , polymerase chain reaction, nucleic acid vaccine, cloning, merozoite surface protein 1
