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。

目的 研究重组人源抗HBsAg单链抗体（HBscFv）在HBV转基因小鼠体内的活性作用，探讨HBV转基因小鼠作为HBsAg特异性抗体的结合活性评价模型的可行性。方法 工程菌表达的HBscFv包涵体经固定化金属螯合层析和分子排阻层析两步纯化后，分步透析复性。复性后的HBscFv（0.9g/L）经尾静脉注射HBV转基因小鼠，一定时间后测定鼠血清中HBsAg的浓度，计算抗体注射前后HBsAg浓度下降的百分比（结合率），同时以人血源HBsAb IgG（40U/ml）和生理盐水作为对照。结果 两步纯化获得纯度达到98%的重组HBscFv。HBscFv制品能够结合转基因小鼠体内的HBsAg，其结合率为（30.47±9.85）%，与生理盐水组差异有显著性（P<0.001），与HBsAb IgG组差异无显著性（P>0.05）；对照品HBsAb IsG的结合率为（39.00±7.43）%，与生理盐水组差异也有显著性（P<0.001）。比较HBscFv和HBsAb IgG的结合率及其浓度，求得HBscFv的结合效价为31.58U/mg。结论 HBscFv在转基因小鼠体内具有特异性结合HBsAg活性，HBV转基因小鼠可发展为评价HBsAs特异性抗体的体内结合活性的候选模型。

目的：探讨灭活SAILS冠状病毒疫苗对小鼠是否有损伤作用。方法：用灭活SARS冠状病毒分别免疫BALB/c和C57BL/6小鼠；ELISA法检测小鼠血清中抗SARS冠状病毒抗体水平；免疫8周后取小鼠各脏器，观察其病理形态学改变。结果：免疫8d后，小鼠血清中就可检测到特异的Ig，M、IgG抗体；组织切片染色观察，可见少数小鼠肺及肝组织出现轻度损伤，其他脏器未见病变。结论：SAILS冠状病毒灭活疫苗可诱导小鼠产生特异性抗体，同时没有造成严重的器官损伤。这将为SARS疫苗进入临床研究打下基础。

Background The etiologic agent of severe acute re spiratory syndrome (SARS) ha s been confirmed to be a novel coronavirus (CoV), namely SARS2CoV1 Developing safe and effective SARS2CoV vaccine s is e ssential for us to prevent the po ssible reemergence of it s epidemic1 Previous experience s indicate that inactivated vaccine is conventional and more hopeful to be succe ssfully developed1 Immunogenicity evaluation of an experimental inactivated SARS2CoV vaccine in rabbit s wa s conducted and reported in this paper1 Methods The large2scale cultured SARS2CoV F69 strain wa s inactivated with 014 % formaldehyde and purified, then used a s the immunogen combined with Freund’s adjuvant1 Eight adult New Zealand rabbit s were immunized four time s with this experimental inactivated vaccine1 Twelve set s of rabbit serum were sampled from the third day to the seventy2fourth day after the first vaccination1 The titers of specific anti-SARS2CoV IgG antibody were determined by indirect enzyme2linked immuno sorbent a ssay, and the neutralizing antibody titers were detected with micro2cytopathic effect neutralization te st1 Results Rapid and potent humoral immune re sponse s were induced by the inactivated SARS2CoV vaccine in all the eight te st rabbit s1 Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1∶81 920 and 1∶20 480,re spectively1 After that, serum antibody levels remained at a plateau or had a slight decrea se, though two boo sters were given in the succedent 4 to 5 weeks1 Cro ss neutralization re sponse existed between SARS2CoV F69 strain and Z22Y3 strain1 Co ncl us i o ns The inactivated SARS2CoV vaccine made from F69 strain owns strong immunogenicity, and the cro ss neutralization re sponse between the two different SARS2CoV strains give s a hint of the similar neutralizing epitope s, which provide stable ba se s for the development of inactivated SARS2 CoV vaccine s1

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.