幽门螺杆菌重组VacA-HpaA IgY的制备

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．

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

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-DH5a was cultured in large numbers to get VacA-HpaA fusion protein. The recombinant protein was purified by Ni2 -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 immu-nosorbent 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.