登革2型病毒E蛋白免疫优势表位的筛选鉴定

目的 用噬菌体展示肽库筛选登革2型病毒(DEN2)E蛋白的抗原表位，并确定该抗原表位性质。方法 以DEN2型特异的E单克隆抗体作为筛选分子，生物淘洗噬菌体随机12肽库，将筛选的噬菌体阳性克隆进行ELISA检测、DNA序列测定及展示肽的氨基酸序列推导，通过噬菌体展示肽序列与DEN2E蛋白的氨基酸一级结构的对比，初步确定E蛋白的抗原表位；用模拟该表位线性序列的合成十肽进行抗体结合试验、噬菌体竞争抑制试验及与DEN感染患者的血清学试验，确定其为免疫优势线性表位。结果 肽库淘洗获得的11个ELISA阳性的噬菌体克隆有相似的结构基序WFKKGSS，其展示肽与DEN2E蛋白390～398 AA序列有3～5个氨基酸相同。对应于DEN2E蛋白390～399AA的合成十肽能与淘洗单抗特异反应，并可抑制噬菌体阳性克隆与该单抗结合。该合成肽与DEN2感染患者血清有较高的免疫反应性。结论 本实验通过噬菌体随机肽库的生物淘洗确定的DEN2E蛋白(E390～398AA)线性序列为免疫优势表位，其对应的合成肽E10可望用于DEN2感染的快速诊断。     

中国狂犬病毒疫苗株（5aG）糖蛋白基因在痘苗病毒中的表达及免疫效果观察

将克隆到的中国狂犬病毒疫苗株（５ａＧ）的糖蛋白基因重组到痘苗病毒ＴＫ区，并在痘苗病毒Ｐ１１启动子的控制下，构建了狂犬－痘苗重组病毒（ＶＶａＧ）。经间接免疫荧光和Ｗｅｓｔｅｒｎ免疫印染证明，重组病毒ＶＶａＧ能良好地表达狂犬病毒糖蛋白，其分子量约为６６００。用ＶＶａＧ免疫小鼠，７ｄ便可诱生较高的狂犬病毒中和抗体，２１ｄ达４１６９，并能１００％保护狂犬病毒本毒株和国际标准攻击毒（ＣＶＳ）的致死量攻击。     

Construction of Canine Herpesvirus Vector Expressing Foreign Genes Using a lacZ-TK Gene Cassette as a Double Selectional Marker

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01–0.1% to 10–80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein). This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the murine interleukin 5 receptor by using a monoclonal antibody

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence Comparison of Avian Infectious Bronchitis Virus S1 Glycoproteins of the Florida Serotype and Five Variant Isolates from Georgia and California

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6–99.3% similar to one another and only 76.6%–76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55–96, 115–149, 255–309, and 378–395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of λgt11 and monoclonal antibodies to map the gene for the 60,000 dalton glycoprotein of infectious laryngotracheitis virus

To localize the gene encoding the 60 kD glycoprotein (gp60) of infectious laryngotracheitis virus (ILTV), a library of the ILTV genome was constructed in the gt11 expression vector. Twelve recombinant bacteriophages expressing gp60 epitopes as fusion products with -galactosidase were detected by immunoscreening with monoclonal antibodies specific for gp60. The ILTV DNA sequence contained in one of these recombinants 24-4 was used as a hybridization probe for mapping the insert sequence on the viral genome. The gene for the gp60 was located at map unit 0.72–0.77 in the unique long region (U_L) of the ILTV genome. The DNA sequence of the 1.2 kb insert of 24-4 containing the gp60 epitope was determined. The majority of deduced gp60 amino acid sequence has no homology with any of the known alphaherpesvirus glycoproteins.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number X 121209.     

Influence of age on serum protein binding of propranolol

Summary The extent of propranolol protein binding was determined in three different age groups of healthy drug-free caucasian males. Volunteers selected for study were 6–15 years old, 25–36 years old and 68–76 years old. Ten milliliters of blood were obtained via venipuncture and collected in glass tubes from the subjects after an overnight fast. Binding determinations were performed by equilibrium dialysis using radiolabelled propranolol. Serum albumin and ₁-acid glycoprotein concentrations were determined in all subjects by radial immunodiffusion. The results obtained showed wide intersubject variability in the binding ratio of propranolol and serum concentrations of ₁-acid glycoprotein. Mean albumin serum concentration was found to be significantly lower in the elderly group as compared to the adult and pediatric groups (p<0.02). A positive correlation was found between the binding ratio of propranolol and the serum concentration of ₁-acid glycoprotein in all the subjects (r=+0.66,p<0.005). No significant correlation was found between the binding ratio of propranolol and the serum concentration of albumin (r=–0.03,p<0.88). These data suggest that the extent of propranolol binding is influenced primarily by serum concentrations of ₁-acid glycoprotein and not by differences in age.     

Drugs affecting coagulation

The clotting cascade is a complex process and is an important survival mechanism. Major haemorrhage and thromboembolic events remain major causes of increased morbidity and mortality. Drugs affecting coagulation have primarily been utilized to treat or reduce the risk of thromboembolic events. However, the recent progress in the management of major trauma and treating coagulopathy has resulted in further research and development of drugs that improve clotting function. Knowledge of drugs used for both clinical circumstances is now required when working in anaesthesia or intensive care.     

非细胞体系核重构过程的超微结构研究

用电镜观察Lambda DNA与非洲爪蟾卵提取物在非细胞体系内的核重构,发现Lambda DNA首先诱导形成类染色质结构,膜泡、核孔复合体围绕这一结构组装成双层核膜,同时类染色质也随着核膜的装配,表现出从致密凝聚到呈现松散均匀分布的变化。有工作表明,在由染色质诱导的核重构过程中,或者是膜泡先与染色质结合,然后有核孔出现,或者是核孔物质先与染色质作用,膜泡通过结合核孔物质形成双层核膜。我们观察由外源DNA诱导的核重构过程,则发现膜泡与核孔复合体先分别独立地与类染色质结构相互作用,然后核孔复合体再镶嵌到双层核膜中。