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.

目的：研究鲨鱼口服液对15名2型糖尿病病人短期病情控制、症状影响。方法：采用单盲、交叉对照等方法，对鲨鱼口服液受试者的血、尿常规，生化指标、血糖及糖化血清蛋白，以及临床症状等指标进行考察，观察时间为20 d。结果：患者在服用鲨鱼口服液可能改善糖尿病的症状，试验前10.7±6.4分，服用口服液后降为5.8±5.3，与安慰剂对比（P<0.05）。同时该试验品对糖尿病患者的肝、肾、心脏功能及血液学功能等无明显不良影响。结论：鲨鱼口服液在2型糖.尿病患者短期应用安全、可能改善症状、应用简便，但对血糖的影响仍需要长期观察。

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

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。

为制备纯化的灭活SARS病毒，取广东省CDC提供的灭活SARS-CoV病谳培养液进行超滤，收集相对分子质量介于100000至500000之间的组分。并浓缩。对纯化过程中各组分进行抗原活性签定和蛋白质含量测定。并加佐剂后免疫动物，测定抗体免疫应答。结果表明：超滤后活性抗原蛋白质回收率7.6%，纯化倍数为9，抗原活性达到1∶480 免疫家免获得的血清ELISA效价达到1∶10000以上，中和抗体效价达到1∶2000以上。表明该纯化工艺能获得较高纯度的灭活SARS-CoV病毒颗粒。

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。