目的：利用中间载体pGEM-T easy vector构建表达型载体pET15b-EGFP，在大肠杆菌中高效表达可溶性蛋白EGFP并将其纯化。方法：以质粒pLEGFP-C1为模板采用聚合酶链反应（PCR）的方法特异性扩增EGFP cD-NA序列，利用Taq DNA聚合酶的非模板依赖性末端转移酶活性催化PCR扩增的EGFP序列和三磷酸脱氧腺苷（dATP）反应，在EGFP序列两3’端加“ ，将纯化后的EGFP序列与中间载体pGEM-T easy vector连接后转化感受态大肠杆菌DH5ct，经蓝白筛选，挑取白色单菌落进行扩增、酶切鉴定，正确重组的质粒命名为pGEM-T-EGFP用XhoI和BamH1分别双酶切pGEM-T-EGFP和pET15b，低熔点琼脂糖回收EGFP eDNA和线性化的pET15b片段后将两片段相连，转化感受态大肠杆菌DH5ct并对重组质粒进行酶切鉴定和DNA测序，获取正确重组的表达型质粒并命名为pET15b-EGFP。将正确重组的质粒pET15b-EGFP转化感受态大肠杆菌BL21（DE3），用异丙基p-D-半乳糖苷（IPTG）诱导表达。利用表达蛋白N端的组氨酸“标签”（His-tag）进行N一树脂柱亲和层析纯化，SDS-PAGE和Western blotting鉴定纯化蛋白质。结杲：经测序证实重组质粒pET15b-EGFP中的EGFP eDNA序列与从Clontech公司购买的质粒pLEGFP-C1中的EGFP eDNA序列完全一致，从而成功构建了表达型重组质粒pET15b-EGFP。pET15b-EGFP转化感受态大肠杆菌BI21（DE ）并经诱导后得到高效表达，表达量可达66me，/100ml菌液，SDS-PAGE和Western blotting显示纯化蛋白为目的蛋白EGFP。结论：表达型重组质粒pET15b-EGFP的成功构建和表达、纯化为细胞穿透肽-EGFP融合蛋白穿透细胞能力的研究奠定了基础。

The immunogenicity of a candidate-inactivated vaccine prepared from SARS-CoV F69 strain was evaluated in Balb/c mice. Potent humoral immune responses were induced under the elicitation of three times of immunizations at 2-week intervals with this vaccine, combined with three types of adjuvants (Freunds adjuvant, Al(OH)3 adjuvant and CpG adjuvant). Titers of specific IgG antibodies in three test groups all peaked in the sixth week after first vaccination, but significant differences existed in the kinetics of specific IgG antibody levels. The strong neutralizing capacity exhibited in micro-cytopathic effect neutralization tests indicated the specific antibodies are protective. Western blot assay further demonstrated the specificity of the induced serum antibodies.

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.

ficial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15[pREP4] respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGF from Origami(DE3) and BL21(DE3) was 1.6μg/L and 2.2μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv] was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from inclusion body in M15[pQE-HBscFv] was 30-35 mg/L and the affinity constant was 1.98×107 mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.