论文部分内容阅读
目的纯化大肠埃希菌Rosetta gami(DE3)表达的可溶性裂殖子表面蛋白1C末端蛋白(MSP1-42,3D7株),检测其体外抑制恶性疟原虫生长的效果。方法利用大肠埃希菌Rosetta gami(DE3)为宿主菌诱导表达MSP1-42,用带组氨酸标签的镍柱纯化可溶性的MSP1-42。6只新西兰白兔随机分为免疫组和对照组,每组3只。用MSP1-42与弗氏佐剂乳化后,皮下注射,抗原免疫剂量每次为200μg/只,共免疫4次,每次间隔2周。佐剂对照组以PBS代替抗原同法免疫。免疫前及末次免疫2周后取血,用酶联免疫吸附试验和间接荧光抗体试验检测血清中特异性抗体及其与天然抗原的反应,用含10%和20%免疫血清的培养基体外培养恶性疟原虫(海南分离株,Fcc1/HN),检测免疫血清体外抑制恶性疟原虫生长的效果。结果经镍柱纯化后获得纯度为95%以上的MSP1-42蛋白;免疫组个体在第4次免疫后血清特异性抗体的滴度依次为1:640000、1:640000、1:160000,间接荧光抗体试验检测表明MSP1-42免疫兔血清与恶性疟原虫表面蛋白有阳性反应;免疫血清能抑制恶性疟原虫在体外生长,在10%浓度时,3只免疫兔血清的抑制率依次为(51.9±24.2)%、(29.4±8.6)%和(86.7±7.4)%,在20%浓度时,抑制率分别为(93.3±7.5)%、(65.3±10.6)%和(96.4±1.0)%。结论大肠埃希菌Rosettagami(DE3)表达的MSP1-42蛋白免疫血清能识别恶性疟原虫天然抗原,体外培养能抑制恶性疟原虫的生长。
Objective To purify soluble mesenchymal stem cell surface protein 1C (MSP1-42, 3D7) expressed by Escherichia coli Rosetta gami (DE3) and test its inhibitory effect on the growth of Plasmodium falciparum in vitro. Methods MSP1-42 was induced by Escherichia coli Rosetta gami (DE3) as a host strain, and purified soluble MSP1-42.6 New Zealand white rabbits with histidine-tagged nickel column were randomly divided into immunization group and control group, 3 per group MSP1-42 and Freund’s adjuvant emulsified, subcutaneous injection, the antigen immunization dose of 200μg / each, a total of four immunizations, each interval of two weeks. Adjuvant control group with PBS instead of antigen immunization. Blood samples were collected two weeks before and after the last immunization. Serum specific antibodies and their reaction with natural antigens were detected by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test, and cultured in vitro with 10% and 20% immune serum medium Plasmodium falciparum (Hainan isolate, Fcc1 / HN) was used to test the effect of immune serum on inhibiting the growth of Plasmodium falciparum in vitro. Results The purity of MSP1-42 was over 95% after purified by Ni-P column. The titer of serum-specific antibody in the immunized group was 1: 640000, 1: 640000, 1: Antibody test showed that MSP1-42 immunized rabbit serum reacted positively with Plasmodium falciparum surface protein. Immune serum could inhibit the growth of Plasmodium falciparum in vitro. At 10% concentration, the inhibitory rates of three immunized rabbits were (51.9 ± 24.2%, 29.4 ± 8.6% and 86.7 ± 7.4%, respectively. The inhibition rates were (93.3 ± 7.5)%, (65.3 ± 10.6)% and (96.4 ± 1.0)% at 20% concentration respectively. Conclusion MSP1-42 protein serum expressed by Escherichia coli Rosettagami (DE3) can recognize the natural antigen of Plasmodium falciparum and inhibit the growth of Plasmodium falciparum in vitro.