论文部分内容阅读
目的建立恶性疟原虫合子/动合子表面25蛋白(25 KDa Plasmodium falciparum sexual stage protein,Pfs25)的ELISA定量检测方法。方法以双抗体夹心ELISA法为基础,建立Pfs25蛋白的ELISA定量检测方法,并对该方法进行重复性和准确性验证。用建立的方法检测不同重组菌株、CHO细胞中,以及经10 L发酵罐培养菌株纯化各阶段样品中Pfs25蛋白的表达量。结果建立的方法线性范围为0~1.6μg/ml,R2=0.984,检测灵敏度为0.032μg/ml,回收率为81%~119.0%,CV﹤15%。Pfs25-Pet42a/DE3表达后的Pfs25蛋白浓度在2.03~7.26μg/ml;Pfs25-p GAPZαA/GS115、Pfs25-p PICZαA/GS115和(α-Pfs25)8-p AO815/GS115表达后的Pfs25蛋白浓度分别为113、105和280μg/ml;3株Pfs25/CHO细胞表达的Pfs25蛋白浓度分别为106.11、116.89和155.01μg/ml。p PICZαAPfs25/GS115菌株发酵液中,Pfs25蛋白的增加呈先快后慢的趋势,72 h后,目的蛋白增加逐渐平缓;纯化洗脱液3个峰中Pfs25蛋白浓度分别为22.67、42.12和55.27μg/ml,流穿液中Pfs25蛋白浓度仅为0.096μg/ml。结论建立的Pfs25蛋白ELISA定量检测方法可用于疟疾疫苗研究中Pfs25蛋白的定量分析。
Objective To establish a method for the quantitative determination of Plasmodium falciparum sexual stage protein 25 (Pfs25) in Plasmodium falciparum. Methods Based on the double antibody sandwich ELISA method, a quantitative ELISA method for the determination of Pfs25 protein was established, and the repeatability and accuracy of the method were verified. The established method was used to detect the expression of Pfs25 protein in various recombinant strains, CHO cells, and the various stages of purification of 10 L fermenter culture strains. Results The linear range of the method was 0 ~ 1.6μg / ml, R2 = 0.984, detection sensitivity was 0.032μg / ml, recovery was 81% ~ 119.0%, CV <15%. Pfs25 protein concentration after Pfs25-Pet42a / DE3 expression was 2.03 ~ 7.26μg / ml; Pfs25 protein concentration after Pfs25-p GAPZαA / GS115, Pfs25-p PICZαA / GS115 and (α-Pfs25) 8-p AO815 / GS115 expression 113, 105 and 280 μg / ml, respectively. The Pfs25 protein concentrations in three Pfs25 / CHO cells were 106.11, 116.89 and 155.01 μg / ml, respectively. The increase of Pfs25 protein in the fermentation broth of PICZαAPfs25 / GS115 showed a tendency of fasting first and then slowing down. After 72 hours, the increase of Pfs25 protein was gradual and gentle. Pfs25 protein concentration in the three peaks of purified eluate was 22.67, 42.12 and 55.27μg / ml, the flowthrough fluid Pfs25 protein concentration of only 0.096μg / ml. Conclusion The established PFS25 protein ELISA assay can be used for the quantitative analysis of Pfs25 protein in malaria vaccine research.