摘要
将恶性疟原虫保护性抗原环子孢子蛋白（ＣＳＰ）、环状体感染红细胞表面抗原（ＲＥＳＡ）、裂殖子表面抗原１和２（ＭＳＡ１和ＭＳＡ２）４种抗原中具有Ｔ和／或Ｂ细胞表位的６个基因片段，按照我们设计的方案排成一条线性序列，经在计算机上分析比较后，分成长短不一的８个基因片段（Ｆ１～Ｆ８），其中Ｆ１、Ｆ３、Ｆ５和Ｆ７为正链片段，Ｆ２、Ｆ４、Ｆ６和Ｆ８为负链片段。每相邻两个片段间预设１５个互补碱基搭头。在ＤＮＡ合成仪上分别合成这８个片段后，把Ｆ２～Ｆ７６个片段分别磷酰化，按等摩尔浓度混合。经３０℃退火，使８个片段连接起来，然后按基因体外扩增的原理，填补互补碱基以外的缺口，重组成新的多价基因。我们把这种化学拼接法取名为“缺口填补法（Ｆｉｌｌｉｎｇ－ｑａｐＭｅｔｈｏｄ）”，经克隆和测序验证，表明这种方法具有效果好、经济、简便等优点。
A 274 bp gene of human plasmodium falciparum hybrid peptide antigen,which isdivided into 8 oligonucleotide fragments(F1～F8),has been synthesized by the solid-phase phos-phoramidite methods with ABI-381A DNA Synthesizer.The gene encodes several T-or B-cellepitopes of CSP,RESA,MSA1 and MSA2.F1,F3,F5,and F7 are located in the positive chain, andF2,F4,F6F8 in the negetive one.Each two neighbouring fragments(i.e ,F1 and F2 ,or F2 and F3)has 15 matching bases.After being annealed and ligated with T4 DNA ligase,the 8 fragments wereinterlinked into a double DNA chain with 8 gaps. Then ,those gaps were filled with the gene am-plification methods. The technique,in combination with genetic chemical splicing and gene ampli-fication in vitro,is named as “Filling-Gap Method”by us.Analysis of PCR or DNA sequencingshows that the filling-gap method is cheaper,simpler and more effective than other methods inrecombing gene.(Fig.1-6)
出处
《中国寄生虫病防治杂志》
CSCD
1994年第4期255-258,共4页
Chinese Journal of Parasitic Disease Control
基金
国家教委博士点基金
卫生部科学基金
关键词
恶性疟原虫
疫苗
缺口填补法
plasmodium falciparum vaccine hybrid peptide antigen filling-gapmethod recombinant gene
