Construction and identification of helper-dependent adenoviral vector encoding hemagglutinin protein of swin-origin influenza virus A H1N1 / 生物医学工程学杂志

Abstract: In order to investigate immune protection against swine-origin influenza virus (S-OIV) A H1N1, the helper-dependent adenovirus vector (HDAd) system was exploited to construct recombinant HDAd encoding hemagglutinin (HA). The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfected with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.

In vitro transgenic expression efficacy of a helper-dependent adenoviral vector encoding enhanced green fluorescent protein / 生物工程学报

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

Isolation and identification of a human single chain Fv antibody against amyloid-beta 1-42 soluble oligomers from a human phage display library / 生物工程学报

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.

Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo.</p><p><b>METHODS</b>The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA. Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot. BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus.</p><p><b>RESULTS</b>Recombinant adenovirus named rvAd-VP6 was obtained. The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably. The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively. In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100. Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group. The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation.</p><p><b>CONCLUSIONS</b>The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.</p>

Developmentof the multiparameter supervisor / 医疗卫生装备

This paper introduces the technical parameters,basic principle and investigation method of the multiparameter supervisor.The functions and implementation methods of its components are also presented.The supervisor can provide much physiological information of the patients to medical staffs.Thus,the level of medical supervision and analysis can be enhanced.The multiparameter supervision can be realized when the microcomputer system with PC-104construction is adopted to analyze the received signals and filter the interfering signals resulting from the random variations of frequency and amplitude.