目的：研究重组工程菌的遗传稳定性。方法：利用重组表达质粒pBVNM-HI转化宿主菌E.coli DI-LSa，筛选重组工程菌DH5a-pBVNM-HI。将新构建好的重组工程菌在无选择压力的条件下进行连续传代培养，比较菌落在LB（-）和LB（+）培养基上的生长状况，并对传代菌株目标蛋白的表达情况以及质粒数量和目的基因DNA进行电泳鉴定。结果：重组工程菌连续传代5O次中，在LB（-）和LB（+）培养基上的生长状况相同，目标蛋白表达量无显著差异，质粒数量及目的基因DNA结构稳定。结论：重组工程菌DH5a-pBVNM.HI具有良好的遗传稳定性。

目的：为提高表达量，简化纯化工艺，获得可用于临床研究的重组人NDPK-A蛋白，构建带有6×His纯化标签的原核表达载体，优化表达条件获得产物高表达并鉴定产物活性。方法：将抑癌基因nm23-H/从质粒pBVNMH1中亚克隆于带有纯化标签的表达载体pQE40中；梯度变化表达条件获得产物高表达；镍离子螯合层析柱纯化目的蛋白；Western blot鉴定产物的免疫原性；HPLC测定产物的激酶活性；鸡胚尿囊膜试验鉴定产物抑制血管新生的生物活性。结果：pQE-40中亚克隆的nm23-H/序列无误；目的蛋白最高表达量可达49.6%；纯化的表达产物能与天然NDPK-A的多克隆抗体特异结合；酶比活为472U/rag；具有抑制鸡胚尿囊膜血管新生的活性。结论：表达质粒-nm23HI能高效表达重组人NDPK-A，纯化工艺简便，产物活性与天然NDPK-A无异。

Severe acute respiratory syndrome (SARS) is a serious infectious threat to public health. To create a novel trial vaccine and evaluate its potency, we attempted to generate a SARS inactivated vaccine using SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)3. Three doses of the vaccine were used to challenge three groups ofBALB/c mice.We found that the mice exhibited specific IgM on day 4 and IgG on day 8. The peak titers of IgG were at day 47 in low-dose group 1∶19,200) and high-dose group (1∶38,400) whereas in middle-dose group (1∶19,200), the peak was at day 40. On day 63, the IgG levels reached a plateau. Neutralization assay demonstrated that the antisera could protect Vero-E6 cells from SARS-CoVs infection. Analysis of the antibody specificity revealed that the mouse antisera contained a mixture of antibodies specifically against the structure proteins of SARS-CoV. Furthermore, the mouse antisera conferred higher amount of antibodies against protein N, polypeptide S4 and S2 than those of proteins M and 3CL. These findings suggest that the inactivated SARS-CoV could preserve its antigenicity and the inactivated vaccine can stimulate mice to produce high levels of ntibodies with neutralization activity. Results also suggest that polypeptides originating from protein N or S might be a potential target for the generation of a recombinant SARS vaccine. © 2004 Elsevier B.V. All rights reserved.