细粒棘球蚴特异性抗原基因P-29的原核表达及免疫学鉴定

Prokaryotic Expression and Immunologic Identification of P-29 Gene from Echinococcus granulosus Hydatid Cyst

目的对细粒棘球蚴P-29基因进行克隆、表达和免疫反应性分析。方法以细粒棘球蚴总RNA为模板,反转录PCR扩增P-29基因,将其克隆至原核表达载体pET44a(+)中,构建重组原核表达载体pET-P-29,转入大肠埃希菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察重组蛋白的表达情况,用蛋白纯化试剂盒纯化蛋白,蛋白质印迹(Western blotting)分析重组蛋白与细粒棘球蚴病患者血清的免疫反应性。结果PCR、双酶切和DNA测序结果表明,重组质粒pET-P-29构建成功。SDS-PAGE结果显示,重组蛋白Nus-P-29的相对分子质量(Mr)约为93000,纯化后的蛋白浓度为0.78mg/ml。重组蛋白Nus-P-29能被细粒棘球蚴病患者血清识别。结论细粒棘球蚴P-29基因表达成功,纯化后的重组蛋白Nus-P-29具有较强的免疫反应性。

Objective To clone and express P-29 gene of Echinococcus granulosus, and analyze its immunore- activity. Methods Total RNA was extracted from the hydatid cyst of E. granulosus and its P-29 gene was amplified by RT-PCR. The PCR product was cloned into pET44a（＋） vector. The recombinant plasmid was transformed into E. coli BL21 （DE3） and followed by expression of the protein induced by IPTG. The protein was purified, and tested by SDS- PAGE. Its immunoreactivity was examined by Western blotting. Results The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant Nus-P-29 was about Mr 93 000 with a concentration of 0.78 mg/ml. It was recognized by the sera of cystic echinococcosis patients. Conclusion The P-29 gene has been expressed with adequate immunoreactivity.