HER-2/neu胞外区的基因工程表达及其免疫避孕作用

Expression of the Extracellular Domain of HER-2/neu and Its Application in Immunocontraception

目的:研究HER-2/neu胞外区部分片段重组蛋白疫苗在免疫避孕中的作用。方法:构建原核表达重组体pET-hECDu,在大肠杆菌中表达人HER-2/neu胞外区部分片段,经Hi-Trap亲和层析柱纯化得到重组蛋白(hECDu),以ELISA法检测其结合活性后免疫雌性小鼠。用ELISA法检测抗HER-2/neu体液免疫应答,3H-TdR掺入法检测细胞免疫应答。结果:经原核表达系统成功纯化得到与理论分子量77 ku(1 u=1 Da)相符的hECDu,并证实其可与抗HER-2/neu胞外区特异性单克隆抗体结合。该重组蛋白疫苗可诱导雌鼠产生较高水平体液及细胞免疫应答。其生育力(平均产仔数4.0±1.8)与对照组(7.8±1.3)相比明显降低,但免疫鼠产后4 d子宫及卵巢均无明显病理改变。结论:经原核表达系统成功纯化得到HER-2/neu胞外区部分片段重组蛋白,该蛋白疫苗可在小鼠中诱导产生免疫避孕效应。

Objectives To study the effects of the recombinant protein vaccine based on the extracellular domain (ECD) of HER-2/neu on immunocontraception. Methods: A carrier pET-hECDu for prokaryotic expression was constructed, the recombinant protein (hECDu) based on ECD was expressed in E. coli and purified by a Hi-Trap Chelating Column (a polyHis-Metal Chelating Affinity Chromatography column). Protein activity was analyzed by ELISA. Female mice were immunized with this vaccine. Specific humoral responses to HER-2/neu were analyzed by ELISA, and specific cellular responses were analyzed by 3H TdR proliferation assay. In addition, contraception effects and histological injuries of uteri were examined. Results:Consistent with the theoretic MW 77ku(1u=1Da), a recombinant protein hECDu was successfully purified by prokaryotic expression system. The protein was confirmed to be able to specifically combine with anti-ECD monoclonal antibody. The high level of humoral and cellular responses was detected in mice immunized with the recombinant protein vaccine. The litter size of the immunized group (average offspring. 4. 0±1. 8) was far less than that of the control group (average offspring:7. 8±1. 3). In comparison with normal mice after delivery, the uteri and ovaries of immunized mice at 4-day after delivery had no obvious pathological change. Conclusion :The recombinant protein vaccine based on ECD could induce immunocontraception in mice.