恶性疟原虫六个保护性抗原表位基因的化学合成及克隆

CHEMICAL SYNTHESIS AND CLONING OF A PLASMODIUM FALCIPARUM HYBRID PEPTIDE ANTIGEN GENE

本文设计并化学合成一条含有恶性疟原虫裂殖子表面抗原（ＭＳＡ１、ＭＳＡ２）、环子孢子蛋白（ＣＳＰ）、环状体感染红细胞表面抗原（ＲＥＳＡ）等不同生活史期的４种抗原，共６个表位的复合基因（ＰｆＣＭＲ）。该基因含２个粘性未端，共分８个片段，采用“缺口填补法”接成双链ＤＮＡ，再与噬菌体Ｍ１３ｍｐ１８分别经ＢａｍＨＩ、ＥｃｏＲＩ双酶切后回收、纯化、重组，并转化ＥｃｏｌｉＪｍ１０９，经ＰＣＲ和酶切鉴定，ＤＮＡ序列分析测定，证实阳性克隆子Ｍ１３ｍｐ１８－Ａ４与预设计基因顺序完全一致。

We have designed and synthesized chemically a hybrid gene encoding several protective epitopes of MSA1,MSA2,RESA in erythrocytic stage antigens and of CSP in pre erythrocytic stage antigen of P.falciparum. The gene with two designed cohesive ends was divided into 8 fragments to be synthesized.All synthetic fragments were annealed and ligated with “fill gap method” to form double DNA chain.This synthetic gene,named PfCMR,was inserted into M13mp18 and transformed into E.coli JM109.The positive recombinants were screened out by PCR and enzyme analysis.The DNA sequence analysis showed that this synthesized gene of hybrid peptide antigens from Plasmadium falciparum was found to be coincided with the sequence of the designed one.