恶性疟原虫杂合45肽抗原基因在减毒鼠伤寒沙门氏菌中的初步表达

Expression of synthetic hybrid 45-peptide gene A of Plasmodium falciparum in attenuated Salmonella typhimurium

将人工合成的恶性疟原虫杂合４５肽基因克隆到表达载体ｐＷＲ４５０－１中，获得了重组质粒ｐＷＲＡ。将ｐＷＲＡ先转化到ＬＢ５０００修饰后，再转化到ＳＬ３２６１，得到重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲＡ）。ｐＷＲＡ在ＳＬ３２６１中以β－半乳糖膏苷酶－杂合多肽抗原Ａ融合蛋白质（ＧＺ－Ａ）形式表达，表达量约７．２％。从ＳＬ３２６１（ｐＷＲＡ）中纯化的ＧＺ－Ａ可与抗ＧＺ－Ａ抗体反应，滴度为１：３２．Ｗｅｓｔｅｒｎｂｌｏｔ显示在ＧＺ－Ａ相应位置出现特异条带。上述结果初步说明ＳＬ３２６１表达的ＧＺ－Ａ具有抗原性。连续传代ＳＬ３２６１（ｐＷＲＡ）未见质粒丢失及明显的毒性，为恶性疟口服活疫苗的制备打下了基础。

synthetic hybrid 45-peptide gene A of plasmodium falciparum was cloned in the expression vectorpWR450-1. The recombinant plasmid pWRA was transformed first into LB5000 to be moidfied , then intoSL3261 to acquire SL3261(pWRA), The recombinant attenuated Salmonella typhimurium SL3261(pWRA)expressed a fusion protein named GZ-A(gene A fused with β-galactosidase(GZ)gene),whichaccounts for about 7.2%of total SL3261 (pWRA) protein. Immunodiffusion assay showed that the puri-fied GZ-A protein from SL3261(pWRA)could react with anti-GZ-A antibody,and the resuIt of Westernblot also showed the specific binding of GZ-A with its antibody. After 53 generations of continuous cul-ture, we found that the recombinant plasmid pWRA was stable in SL3261 and had no obvious toxicity. Allof these may have brotight the appearance of P falciparum oral vaccine a step nearer. More work are underwavnow.