幽门螺杆菌重组VacA-HpaA IgY的制备

Preparation of egg yolk immunoglubin against recombinant vacuolating cytotoxin A-Helicobacter pylori adhesin A in Helicobacter pylori

目的:采用基因工程技术大量表达、提纯幽门螺杆菌(H pylori)细胞空泡毒素毒性片段,和黏附素片段的融合蛋白,以此为抗原,制备高效价的抗VacA-HpaA蛋黄抗体(IgY)．方法:大量培养重组菌pQE30-VacA-HpaA- DH5α,诱导表达获得融合蛋白VacA—HpaA, Ni2+-NTA树脂纯化．将纯化的重组蛋白免疫鸡,水稀释法提取IgY,硫酸铵沉淀法纯化浓缩VacA—HpaA IgY．ELISA法测定抗体产生的时间-效价变化,SDS—PAGE分析纯度, Bradford法测定蛋白含量,Western blot检测其分别对VacA和HpaA抗原的特异性,ELISA法检测效价．结果:重组蛋白主要以包涵体形式表达,免疫鸡后提取的IgY可与该蛋白发生免疫反应, IgY效价总体上随免疫时间增加而升高．经纯化、浓缩后,获得纯度为60％左右,效价为1: 12 800的VacA-HpaA IgY,蛋白浓度为22 g／L, Westem blot显示在Mr27 000和Mr30 000处分别有相应条带,ELISA检测与VacA和HpaA反应的效价分别为1:3200和1:6400(P<0．01)．结论:成功制备了高浓度、高效价的抗重组VacA-HpaA的特异性IgY．

AIM： To prepare a highly specific and efficient egg yolk immunoglubin （IgY） against recombinant vacuolating cytotoxin A-Helicobacter pylori adhesin A （VacA-HpaA） from the yolk of hen＇s eggs.METHODS： Recombinant bacteria of pQE30- VacA-HpaA-DH5α was cultured in large numbers to get VacA-HpaA fusion protein. The recombinant protein was purified by Ni^2＋-NTA column chromatography and used to immunize the hens. The VacA-HpaA IgY was extracted from the yolk of hen＇s eggs by water-dilution methods. In order to evaluate the relationship between IgY titer and immune time, the titer of IgY was detected by enzyme-linked immunosorbent assay （ELISA）. IgY was purified and concentrated by deposition technique with ammonium sulfate. The purity of IgY was analyzed by SDS-PAGE, and protein content of IgY was checked by Bradford method. The specificities of VacA-HpaA IgY to the antigens of VacA and HpaA were identified by Western blotting. RESULTS： The recombinant protein was mainly expressed as inclusion body. The content of fusion protein was 0.72 g/L. VacA-HpaA IgY from eggs＇ yolk of hens immunized with the fusion protein could react with the fusion protein. The titer of VacA-HpaA IgY was increased with the immune time. After purification and concentration, the purity of VacA-HpaA IgY was about 60%; the titer was 1 ： 128 000; And the concentration of IgY was 22 g/L. Western blot exhibited the protein bands with molecular weight of 27 000 and 30 000. The titer of VacA-HpaA IgY to VacA and HpaA were 1：3200 and 1： 6400 （P 〈 0.01）.CONCLUSION： VacA-HpaA-IgY with high concentration, purity, and specificity is successfully prepared.