卡氏肺孢子虫p55抗原基因片段原核表达质粒的构建与表达

目的 构建卡氏肺孢子虫p55抗原基因片段原核重组表达质粒，表达重组蛋白。 方法 SD大鼠皮下注射免疫抑制剂地塞米松14周，建立卡氏肺孢子虫大鼠模型。从感染大鼠肺组织中抽提总RNA，RT-PCR克隆p55基因，测序, 验证扩增产物。利用定向克隆技术将p55基因片段克隆到载体pGEX-4T-1上，重组质粒经酶切分析、PCR鉴定后，用异丙基?鄄β?鄄D-硫代半乳糖苷(IPTG)诱导表达。最后用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE) 和蛋白质印迹（Western blotting）分析鉴定表达产物。 结果 克隆的p55基因片段为690 bp, 重组质粒pGEX-4T-1/690构建成功； SDS-PAGE显示目的蛋白相对分子质量(Mr)约为62 000，表达产量约占菌体蛋白的11.6%；Western blotting分析结果显示，表达蛋白能分别与谷胱甘肽（GST）抗体、卡氏肺孢子虫感染的大鼠血清发生反应。 结论 构建了卡氏肺孢子虫p55抗原基因的pGEX-4T-1/690重组质粒，诱导表达的蛋白具有良好的抗原性。

Construction and Expression of Prokaryotic Expression Plasmids of Pneumocystis carinii p55 Antigen Gene Fragment

OBJECTIVE: To construct prokaryotic recombinant expression plasmid carrying Pneumocystis carinii Mr 55 000 antigen (p55) gene fragment and express the recombinant protein. METHODS: P. carinii pneumonia (PcP) rat models were established by subcutaneous injection of dexamethasone for 14 weeks. Total RNA was extracted from lung of P. carinii rat and p55 antigen gene fragment was cloned by RT-PCR, which was identified by sequencing. The 690 bp fragment was cloned to pGEX-4T-1, the recombinant plasmid was screened and identified by restriction analysis and PCR. The recombinant plasmid was finally induced with IPTG to express a new fusion protein, and the products were analyzed by SDS-PAGE and Western blot. RESULTS: A fragment of 690 bp was obtained by RT-PCR. The recombinant pGEX-4T-1/690 was constructed. SDS-PAGE revealed that the molecular weight of the recombinant protein was approximately Mr 62,000, the maximum amount of the fusion protein produced was 11.6% of the total protein. The recombinant protein can be recognized by GST antibody and by the sera from P. carinii infected rats using Western blotting. CONCLUSION: Prokaryotic expression plasmid pGEX-4T-1/690 has been constructed and the recombinant fusion protein shows antigenicity.