恶性疟原虫疫苗研究Ⅳ──恶性疟原虫保护性抗原复合基因(PfCMR)的克隆与高效表达

Study on Plasmodium falciparum Vaccine Ⅳ Cloning and High Level Expression of Hybrid Gene Encodiug Protective Antigens of P.falciparum

将恶性疟原虫保护性抗原复合基因（PfCMR）与表达质粒pWR450—1分别经BamHI、EcoRI双酶切后回收、纯化和重组并转化大肠杆菌JM109，再经含氨苄青霉素LB培基初筛后，挑白色菌落扩增，用PCR复筛和双酶切鉴定，阳性克隆子pWR450—1—B14用IPTG诱导，在JM109中表达。表达产物经SDS—PAGE分析，在65kDa处出现较宽的蛋白条带。用SOS显色法证实此表达蛋白具有β-半乳糖苷酶活性。表达的融合蛋白与抗恶性疟原虫多克隆免疫血清特异结合．其滴度高达1：3200；Westernblot分析显示，表达蛋白能与特异性抗体产生较强的免疫反应。上述结果提示表达的融合蛋白具有生物学和免疫学活性。

By a serial methods of recovery, purification, recombination, the recombinant vector pWR450-1-B14 wasconstructed. After transformation with pWR450-1-B14,the genetically engineered bacteria were induced to beexpressed with IPTG. The product was analyzed by SDS-PAGE. The result showed that a novel wide bandcould be seen at 65KDa in PAGE. The expressed protein was also proved to exhibit the activity of β-galactosidase by SOS method. In addition,the immunogenicity of the product was observed using ELISA and Western blotanalyses. The results demonstrated that the expressed protein strongly reacted with immune rabbit serum againstP. falciparum. The titer was up to 1: 3200 as detected by ELISA. These findings suggested that the productcould possess both the activity of β-galactosidase and the immunological specificity of P. falciparam antigens.